Tricyclic compounds as inhibitors of kras

ABSTRACT

Disclosed are compounds of Formula I, methods of using the compounds for inhibiting KRAS activity and pharmaceutical compositions comprising such compounds. The compounds are useful in treating, preventing or ameliorating diseases or disorders associated with KRAS activity such as cancer.

RELATED APPLICATIONS

This application is related to U.S. Provisional Application No.63/219,274 filed Jul. 7, 2021; U.S. Provisional Application No.63/292,774, filed Dec. 22, 2021; and U.S. Provisional Application No.63/310,811, filed Feb. 16, 2022, the contents of which are incorporatedin their entireties.

FIELD OF THE INVENTION

The disclosure provides compounds as well as their compositions andmethods of use. The compounds modulate KRAS activity and are useful inthe treatment of various diseases including cancer.

BACKGROUND OF THE INVENTION

Ras proteins are part of the family of small GTPases that are activatedby growth factors and various extracellular stimuli. The Ras familyregulates intracellular signaling pathways responsible for growth,migration, survival and differentiation of cells. Activation of RASproteins at the cell membrane results in the binding of key effectorsand initiation of a cascade of intracellular signaling pathways withinthe cell, including the RAF and PI3K kinase pathways. Somatic mutationsin RAS may result in uncontrolled cell growth and malignanttransformation while the activation of RAS proteins is tightly regulatedin normal cells (Simanshu, D. et al. Cell 170.1 (2017):17-33).

The Ras family is comprised of three members: KRAS, NRAS and HRAS. RASmutant cancers account for about 25% of human cancers. KRAS is the mostfrequently mutated isoform accounting for 85% of all RAS mutationswhereas NRAS and HRAS are found mutated in 12% and 3% of all Ras mutantcancers respectively (Simanshu, D. et al. Cell 170.1 (2017):17-33). KRASmutations are prevalent amongst the top three most deadly cancer types:pancreatic (97%), colorectal (44%), and lung (30%) (Cox, A. D. et al.Nat Rev Drug Discov (2014) 13:828-51). The majority of RAS mutationsoccur at amino acid residue 12, 13, and 61. The frequency of specificmutations varies between RAS gene isoforms and while G12 and Q61mutations are predominant in KRAS and NRAS respectively, G12, G13 andQ61 mutations are most frequent in HRAS. Furthermore, the spectrum ofmutations in a RAS isoform differs between cancer types. For example,KRAS G12D mutations predominate in pancreatic cancers (51%), followed bycolorectal adenocarcinomas (45%) and lung cancers (17%) while KRAS G12 Vmutations are associated with pancreatic cancers (30%), followed bycolorectal adenocarcinomas (27%) and lung adenocarcinomas (23%) (Cox, A.D. et al. Nat Rev Drug Discov (2014) 13:828-51). In contrast, KRAS G12Cmutations predominate in non-small cell lung cancer (NSCLC) comprising11-16% of lung adenocarcinomas, and 2-5% of pancreatic and colorectaladenocarcinomas (Cox, A. D. et al. Nat. Rev. Drug Discov. (2014)13:828-51). Genomic studies across hundreds of cancer cell lines havedemonstrated that cancer cells harboring KRAS mutations are highlydependent on KRAS function for cell growth and survival (McDonald, R. etal. Cell 170 (2017): 577-592). The role of mutant KRAS as an oncogenicdriver is further supported by extensive in vivo experimental evidenceshowing mutant KRAS is required for early tumour onset and maintenancein animal models (Cox, A. D. et al. Nat Rev Drug Discov (2014)13:828-51).

Taken together, these findings suggest that KRAS mutations play acritical role in human cancers; development of inhibitors targetingmutant KRAS may therefore be useful in the clinical treatment ofdiseases that are characterized by a KRAS mutation.

SUMMARY

The present disclosure provides, inter alia, a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein constituentvariables are defined herein.

The present disclosure further provides a pharmaceutical compositioncomprising a compound of the disclosure, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

The present disclosure further provides methods of inhibiting KRASactivity, which comprises administering to an individual a compound ofthe disclosure, or a pharmaceutically acceptable salt thereof. Thepresent disclosure also provides uses of the compounds described hereinin the manufacture of a medicament for use in therapy. The presentdisclosure also provides the compounds described herein for use intherapy.

The present disclosure further provides methods of treating a disease ordisorder in a patient comprising administering to the patient atherapeutically effective amount of a compound of the disclosure, or apharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION Compounds

In an aspect, provided herein is a compound having Formula (I

or a pharmaceutically acceptable salt thereof, wherein:

Y is N or CH;

R¹ is selected from Cl, CH₃, CH₂F, CHF₂, and CF₃;

Cy¹ is selected from

R² is selected from F and Cl;

R³ is selected from

and

Cy² is selected from

provided that the compound of Formula I is other than,

2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile,2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile, and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile.

In an embodiment of Formula I, or a pharmaceutically acceptable saltthereof

Y is N or CH;

R¹ is selected from Cl, CH₃, H_(Z)F, CHF₂, and CF₃;

Cy¹ is selected from

R² is selected from F and Cl;

R³ is selected from

and

Cy² is selected from

provided that the compound of Formula I is other than,

2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile.

In yet another embodiment, the compound of Formula I is a compound ofFormula II:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from Cl and CH₃;

Cy¹ is selected from

R³ is selected from

and

Cy² is selected from

In an embodiment of Formula I, or a pharmaceutically acceptable saltthereof,

Y is N or CH;

R¹ is selected from Cl, OH₃, CH₂F, CHF₂, and OF₃;

Cy¹ is selected from

R² is selected from F and Cl;

R³ is selected from

and,

Cy² is selected from

In an embodiment, Y is CH. In an embodiment Y is N.

In an embodiment, Cy¹ is selected from Cy¹-c, Cy¹-l, Cy¹-m, Cy¹-n,Cy¹-o, Cy¹-p, Cy¹-q, Cy¹-r, Cy¹-s, and Cy¹-t. In an embodiment, Cy¹ isselected from Cy¹-l, Cy¹-m, Cy¹-n, Cy¹-o, Cy¹-p, Cy¹-q, Cy¹-r, Cy¹-s,and Cy¹-t. In an embodiment, Cy¹ is selected from Cy¹-c, Cy¹-m, Cy¹-n,Cy¹-o, Cy¹-p, Cy¹-q, Cy¹-r, Cy¹-s, and Cy¹-t. In an embodiment, Cy¹ isselected from Cy¹-f, Cy¹-g, Cy¹-h, Cy¹-i, Cy¹-j, Cy¹-k, and Cy¹-l. In anembodiment, Cy¹ is selected from Cy¹-a, Cy¹-m, Cy¹-n, Cy¹-o, Cy¹-p, andCy¹-q. In an embodiment, Cy¹ is selected from Cy¹-c, Cy¹-d, Cy¹-e,Cy¹-r, Cy¹-s, and Cy¹-t.

In an embodiment, Cy¹ is selected from Cy¹-a, Cy¹-c, and Cy¹-r. In anembodiment, Cy¹ is selected from Cy¹-c and Cy¹-r. In an embodiment, Cy¹is selected from Cy¹-s and Cy¹-t.

In an embodiment, Cy¹ is Cy¹-l. In an embodiment, Cy¹ is Cy¹-m. In anembodiment, Cy¹ is Cy¹-n. In an embodiment, Cy¹ is Cy¹-o. In anembodiment, Cy¹ is Cy¹-p. In an embodiment, Cy¹ is Cy¹-q. In anembodiment, Cy¹ is Cy¹-r. In an embodiment, Cy¹ is Cy¹-s.

In an embodiment, Cy¹ is Cy¹-t.

In an embodiment, Cy¹ is selected from Cy¹-a, Cy¹-b, Cy¹-c, Cy¹-d,Cy¹-e, Cy¹-f, Cy¹-g, Cy¹-i, and Cy¹-j. In an embodiment, Cy¹ is selectedfrom Cy¹-a, Cy¹-b, Cy¹-c, Cy¹-d, and Cy¹-e. In an embodiment, Cy¹ isselected from Cy¹-f, Cy¹-g, Cy¹-h, Cy¹-i, Cy¹-j, and Cy¹-k. In anembodiment, Cy¹ is selected from Cy¹-c, Cy¹-d, and Cy¹-e. In anembodiment, Cy¹ is selected from Cy¹-a, Cy¹-b, and Cy¹-k. In anembodiment, Cy¹ is selected from Cy¹-a and Cy¹-b. In an embodiment, Cy¹is selected from Cy¹-h and Cy¹-k.

In an embodiment, Cy¹ is Cy¹-a. In an embodiment, Cy¹ is Cy¹-b. In anembodiment, Cy¹ is Cy¹-c. In an embodiment, Cy¹ is Cy¹-d. In anembodiment, Cy¹ is Cy¹-e. In an embodiment, Cy¹ is Cy¹-f. In anembodiment, Cy¹ is Cy¹-g. In an embodiment, Cy¹ is Cy¹-h. In anembodiment, Cy¹ is Cy¹-i. In an embodiment, Cy¹ is Cy¹-j. In anembodiment, Cy¹ is Cy¹-k.

In an embodiment, R¹ is selected from CH₃, CH₂F, CHF₂, and CF₃. In anembodiment, R¹ is selected from Cl, CH₃, CHF₂, and CF₃. In anembodiment, R¹ is selected from CH₂F, CHF₂, and CF₃. In an embodiment,R¹ is selected from Cl, CH₂F, CHF₂, and CF₃. In an embodiment, R¹ isselected from Cl and CH₃. In an embodiment, R¹ is selected from C1 andCF₃. In an embodiment, R¹ is selected from CH₃ and CF₃. In anembodiment, R¹ is C1. In an embodiment, R¹ is CH₂F. In an embodiment, R¹is CHF₂. In an embodiment, R¹ is CH₃. In an embodiment, R¹ is CF₃.

In an embodiment, R² is F. In an embodiment R² is Cl.

In an embodiment, R³ is selected from R³-a and R³-b. In an embodiment,R³ is selected from R³-b and R³-c. In an embodiment, R³ is selected fromR³-a and R³-c. In an embodiment, R³ is R³-a. In an embodiment, R³ isR³-b. In an embodiment, R³ is R³-c. In an embodiment, R³ is selectedfrom R³-b, R³-c, and R³-d. In an embodiment, R³ is selected from R³-b,and R³-d. In an embodiment, R³ is selected from R³-c, and R³-d. In anembodiment, R³ is selected from R³-a, and R³-d. In an embodiment, R³ isR³-d.

In an embodiment, Cy² is selected from Cy²-b, Cy²-d, Cy²-e, and Cy²-f.In an embodiment, Cy² is selected from Cy²-b and Cy²-d. In anembodiment, Cy² is selected from Cy²-c and Cy²-d.

In an embodiment, Cy² is selected from Cy²-a, Cy²-c, and Cy²-d. In anembodiment, Cy² is selected from Cy²-a, Cy²-b, and Cy²-d. In anembodiment, Cy² is selected from Cy²-a, Cy²-b, and Cy²-c. In anembodiment, Cy² is selected from Cy²-d, Cy²-e, and Cy²-f. In anembodiment, Cy² is selected from Cy²-a and Cy²-b. In an embodiment, Cy²is selected from Cy²-c and Cy²-f. In an embodiment, Cy² is selected fromCy²-e and Cy²-f.

In an embodiment, Cy² is Cy²-a. In an embodiment, Cy² is Cy²-b. In anembodiment, Cy² is Cy²-c. In an embodiment, Cy² is Cy²-d. In anembodiment, Cy² is Cy²-e. In an embodiment, Cy² is Cy²-f.

In another embodiment, the compound of Formula I is other than,

2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile,2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile, and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile.

In an embodiment, the compound of Formula I is other than,

2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile.

In another embodiment, compounds of the Formulae herein are compounds ofthe Formulae or pharmaceutically acceptable salts thereof.

In an embodiment, the compound of Formula I is selected from

-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   1-(4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-1-yl)prop-2-en-1-one;-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(4-fluorophenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   8-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   8-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   8-(6-fluoro-1-(1-((E)-4-fluorobut-2-enoyl)piperidin-4-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   8-(1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)    piperidin-2-yl)acetonitrile;    2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;    and    2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;    -   or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula I is selected from

-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   1-(4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-1-yl)prop-2-en-1-one;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)    piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)    piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)    piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(4-fluorophenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   8-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   8-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   8-(6-fluoro-1-(1-((E)-4-fluorobut-2-enoyl)piperidin-4-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   8-(1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;-   2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)    piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;    and-   2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)    piperidin-2-yl)acetonitrile;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula I is selected from:

-   2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indazol-3-yl)acetonitrile;-   2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile;-   2-(4-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile;-   2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;    and-   2-((2S,4S)-4-(8-chloro-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;

or a pharmaceutically acceptable salt thereof.

In an embodiment, the compound of Formula I is selected from

-   2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;-   2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;

or a pharmaceutically acceptable salt thereof.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment (while theembodiments are intended to be combined as if written in multiplydependent form). Conversely, various features of the invention whichare, for brevity, described in the context of a single embodiment, canalso be provided separately or in any suitable subcombination. Thus, itis contemplated as features described as embodiments of the compounds ofFormula I can be combined in any suitable combination.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically inactive startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis. Many geometric isomers ofolefins, C═N double bonds and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. One method includes fractionalrecrystallization using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, e.g., optically active acids,such as the D and L forms of tartaric acid, diacetyltartaric acid,dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or thevarious optically active camphorsulfonic acids such as β-camphorsulfonicacid. Other resolving agents suitable for fractional crystallizationmethods include stereoisomerically pure forms of α-methylbenzylamine(e.g., S and R forms, or diastereomerically pure forms),2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

In some embodiments, the compounds of the invention have the(R)-configuration. In other embodiments, the compounds have the(S)-configuration. In compounds with more than one chiral centers, eachof the chiral centers in the compound may be independently (R) or (S),unless otherwise indicated.

Compounds of the invention also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone-enol pairs, amide-imidic acidpairs, lactam-lactim pairs, enamine-imine pairs, and annular forms wherea proton can occupy two or more positions of a heterocyclic system,e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium. One ormore constituent atoms of the compounds of the invention can be replacedor substituted with isotopes of the atoms in natural or non-naturalabundance. In some embodiments, the compound includes at least onedeuterium atom. For example, one or more hydrogen atoms in a compound ofthe present disclosure can be replaced or substituted by deuterium. Insome embodiments, the compound includes two or more deuterium atoms. Insome embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 deuterium atoms. Synthetic methods for including isotopes intoorganic compounds are known in the art (Deuterium Labeling in OrganicChemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts,1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau,Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007,7744-7765; The Organic Chemistry of Isotopic Labelling by James R.Hanson, Royal Society of Chemistry, 2011). Isotopically labeledcompounds can used in various studies such as NMR spectroscopy,metabolism experiments, and/or assays.

Substitution with heavier isotopes such as deuterium, may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence may be preferred in some circumstances. (A. Kerekes et. al. J.Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm.2015, 58, 308-312).

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, tautomers and isotopes of thestructures depicted. The term is also meant to refer to compounds of theinventions, regardless of how they are prepared, e.g., synthetically,through biological process (e.g., metabolism or enzyme conversion), or acombination thereof.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates) or can be isolated. When in the solid state, thecompounds described herein and salts thereof may occur in various formsand may, e.g., take the form of solvates, including hydrates. Thecompounds may be in any solid state form, such as a polymorph orsolvate, so unless clearly indicated otherwise, reference in thespecification to compounds and salts thereof should be understood asencompassing any solid state form of the compound.

In some embodiments, the compounds of the invention, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, e.g., a composition enriched in the compounds of the invention.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compounds of the invention, or salt thereof.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g., a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, e.g., a temperature from about 20°C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. The term “pharmaceutically acceptablesalts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the present invention include the non-toxic salts of the parentcompound formed, e.g., from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, alcohols (e.g., methanol, ethanol,iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J.Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook ofPharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). Insome embodiments, the compounds described herein include the N-oxideforms.

Synthesis

Compounds of the invention, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, such as those inthe Schemes below.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediatesor products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups is described, e.g., in Kocienski, Protecting Groups,(Thieme, 2007); Robertson, Protecting Group Chemistry, (OxfordUniversity Press, 2000); Smith et al., March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley,2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,”J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groupsin Organic Synthesis, 4th Ed., (Wiley, 2006).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry or by chromatographic methods such as high-performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

The Schemes below provide general guidance in connection with preparingthe compounds of the invention. One skilled in the art would understandthat the preparations shown in the Schemes can be modified or optimizedusing general knowledge of organic chemistry to prepare variouscompounds of the invention.

Compounds of formula 1-18 can be prepared via the synthetic routeoutlined in Scheme 1. Halogenation of starting material 1-1 with anappropriate reagent, such as N-chloro-succinimide (NCS), affordsintermediate 1-2 (Hal is a halide, such as F, Cl, Br, or I). Compound1-3 can be prepared by treating 1-2 with reagents such as triphosgene.Intermediate 1-3 can then react with ester 1-4 to deliver the nitrocompound 1-5, which can be treated with an appropriate reagent (e.g.,POCl₃) to afford compound 1-6. A S_(N)Ar reaction of intermediate 1-6with amine 1-7 (PG is an appropriate protecting group, such as Boc) canbe carried out to generate compound 1-8. The nitro group in 1-8 can bereduced to NH₂ in the presence of reducing agents (e.g., Fe in aceticacid or sodium dithionite). Intermediate 1-9 can then undergo acyclization reaction (e.g., using triethyl orthoformate) to yieldintermediate 1-10, followed by a SnAr reaction with sodium thiomethoxideto provide 1-11. A cross-coupling reaction with 1-12, in which M is aboronic acid, boronic ester or an appropriately substituted metal ormetalloid [e.g., M is B(OR)₂, Sn(Alkyl)₃, Zn-Hal, or CF₃TMS], understandard Suzuki Cross-Coupling conditions (e.g., in the presence of apalladium catalyst and a suitable base), or standard Stillecross-coupling conditions (e.g., in the presence of a palladiumcatalyst), or standard Negishi cross-coupling conditions (e.g., in thepresence of a palladium catalyst), or trifluoromethylation conditions(e.g., in the presence of a copper catalyst) yields 1-13. Intermediate1-15 can be prepared by a cross coupling reaction between 1-13 and anadduct of formula 1-14, in which M is a boronic acid, boronic ester oran appropriately substituted metal [e.g., M is B(OR)₂, Sn(Alkyl)₃, orZn-Hal], under standard Suzuki Cross-Coupling conditions (e.g., in thepresence of a palladium catalyst and a suitable base), or standardStille cross-coupling conditions (e.g., in the presence of a palladiumcatalyst), or standard Negishi cross-coupling conditions (e.g., in thepresence of a palladium catalyst). Intermediate 1-15 can be converted tointermediate 1-16 either through oxidation of the sulfur group with asuitable oxidant (e.g. m-CPBA) followed by an SnAr reaction, or across-coupling reaction (Org. Lett. 2002, 4, 979-981). Removal of theprotecting group in 1-16 affords the amine 1-17. Functionalization ofthe resulting amine (such as coupling with acid chloride, e.g. acryloylchloride) then affords the desired product 1-18. The order of the abovedescribed chemical reactions can be rearranged or omitted as appropriateto suit the preparation of different analogues.

KRAS Protein

The Ras family is comprised of three members: KRAS, NRAS and HRAS. RASmutant cancers account for about 25% of human cancers. KRAS is the mostfrequently mutated isoform in human cancers: 85% of all RAS mutationsare in KRAS, 12% in NRAS, and 3% in HRAS (Simanshu, D. et al. Cell 170.1(2017):17-33). KRAS mutations are prevalent amongst the top three mostdeadly cancer types: pancreatic (97%), colorectal (44%), and lung (30%)(Cox, A. D. et al. Nat Rev Drug Discov (2014) 13:828-51). The majorityof RAS mutations occur at amino acid residues/codons 12, 13, and 61;Codon 12 mutations are most frequent in KRAS. The frequency of specificmutations varied between RAS genes and G12D mutations are mostpredominant in KRAS whereas Q61R and G12R mutations are most frequent inNRAS and HRAS. Furthermore, the spectrum of mutations in a RAS isoformdiffers between cancer types. For example, KRAS G12D mutationspredominate in pancreatic cancers (51%), followed by colorectaladenocarcinomas (45%) and lung cancers (17%) (Cox, A. D. et al. Nat RevDrug Discov (2014) 13:828-51). In contrast, KRAS G12C mutationspredominate in non-small cell lung cancer (NSCLC) comprising 11-16% oflung adenocarcinomas (nearly half of mutant KRAS is G12C), as well as2-5% of pancreatic and colorectal adenocarcinomas, respectively (Cox, A.D. et al. Nat. Rev. Drug Discov. (2014) 13:828-51). Using shRNAknockdown thousands of genes across hundreds of cancer cell lines,genomic studies have demonstrated that cancer cells exhibiting KRASmutations are highly dependent on KRAS function for cell growth(McDonald, R. et al. Cell 170 (2017): 577-592). Taken together, thesefindings suggested that KRAS mutations play a critical role in humancancers, therefore development of the inhibitors targeting mutant KRASmay be useful in the clinical treatment of diseases that havecharacterized by a KRAS mutation.

METHODS OF USE

The cancer types in which KRAS harboring G12C, G12V and G12D mutationsare implicated include, but are not limited to: carcinomas (e.g.,pancreatic, colorectal, lung, bladder, gastric, esophageal, breast, headand neck, cervical skin, thyroid); hematopoietic malignancies (e.g.,myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS),chronic and juvenile myelomonocytic leukemia (CMML and JMML), acutemyeloid leukemia (AML), acute lymphocytic leukemia (ALL) and multiplemyeloma (MM)); and other neoplasms (e.g., glioblastoma and sarcomas). Inaddition, KRAS mutations were found in acquired resistance to anti-EGFRtherapy (Knickelbein, K. et al. Genes & Cancer, (2015): 4-12). KRASmutations were found in immunological and inflammatory disorders(Fernandez-Medarde, A. et al. Genes & Cancer, (2011): 344-358) such asRas-associated lymphoproliferative disorder (RALD) or juvenilemyelomonocytic leukemia (JMML) caused by somatic mutations of KRAS orNRAS.

Compounds of the present disclosure can inhibit the activity of the KRASprotein. For example, compounds of the present disclosure can be used toinhibit activity of KRAS in a cell or in an individual or patient inneed of inhibition of the enzyme by administering an inhibiting amountof one or more compounds of the present disclosure to the cell,individual, or patient.

As KRAS inhibitors, the compounds of the present disclosure are usefulin the treatment of various diseases associated with abnormal expressionor activity of KRAS. Compounds which inhibit KRAS will be useful inproviding a means of preventing the growth or inducing apoptosis intumors, or by inhibiting angiogenesis. It is therefore anticipated thatcompounds of the present disclosure will prove useful in treating orpreventing proliferative disorders such as cancers. In particular,tumors with activating mutants of receptor tyrosine kinases orupregulation of receptor tyrosine kinases may be particularly sensitiveto the inhibitors.

In an aspect, provided herein is a method of inhibiting KRAS activity,said method comprising contacting a compound of the instant disclosurewith KRAS. In an embodiment, the contacting comprises administering thecompound to a patient.

In an aspect, provided herein is a method of inhibiting a KRAS proteinharboring a G12C mutation, said method comprising contacting a compoundof the instant disclosure with KRAS.

In an aspect, provided herein is a method of inhibiting a KRAS proteinharboring a G12D mutation, said method comprising contacting a compoundof the instant disclosure with KRAS.

In an aspect, provided herein is a method of inhibiting a KRAS proteinharboring a G12V mutation, said method comprising contacting a compoundof the instant disclosure with KRAS.

In another aspect, provided herein a is method of treating a disease ordisorder associated with inhibition of KRAS interaction, said methodcomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of any of the formulae disclosed herein,or pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a method of treating a diseaseor disorder associated with inhibiting a KRAS protein harboring a G12Cmutation, said method comprising administering to a patient in needthereof a therapeutically effective amount of a compound of any of theformulae disclosed herein, or pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is also a method of treatingcancer in a patient in need thereof comprising administering to thepatient a therapeutically effective amount of the compounds disclosedherein wherein the cancer is characterized by an interaction with a KRASprotein harboring a G12C mutation.

In yet another aspect, provided herein is a method for treating a cancerin a patient, said method comprising administering to the patient atherapeutically effective amount of any one of the compounds disclosedherein, or pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a disease ordisorder associated with inhibition of KRAS interaction or a mutantthereof, in a patient in need thereof, comprising the step ofadministering to the patient a compound disclosed herein, or apharmaceutically acceptable salt thereof, or a composition comprising acompound disclosed herein or a pharmaceutically acceptable salt thereof,in combination with another therapy or therapeutic agent as describedherein.

In an embodiment, the cancer is selected from hematological cancers,sarcomas, lung cancers, gastrointestinal cancers, genitourinary tractcancers, liver cancers, bone cancers, nervous system cancers,gynecological cancers, and skin cancers.

In another embodiment, the lung cancer is selected from non-small celllung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma,squamous cell bronchogenic carcinoma, undifferentiated small cellbronchogenic carcinoma, undifferentiated large cell bronchogeniccarcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma,bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma,mesothelioma, pavicellular and non-pavicellular carcinoma, bronchialadenoma, and pleuropulmonary blastoma.

In yet another embodiment, the lung cancer is non-small cell lung cancer(NSCLC). In still another embodiment, the lung cancer is adenocarcinoma.

In an embodiment, the gastrointestinal cancer is selected from esophagussquamous cell carcinoma, esophagus adenocarcinoma, esophagusleiomyosarcoma, esophagus lymphoma, stomach carcinoma, stomach lymphoma,stomach leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductaladenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma,pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma,small bowel adenocarcinoma, small bowel lymphoma, small bowel carcinoidtumors, Kaposi's sarcoma, small bowel leiomyoma, small bowel hemangioma,small bowel lipoma, small bowel neurofibroma, small bowel fibroma, largebowel adenocarcinoma, large bowel tubular adenoma, large bowel villousadenoma, large bowel hamartoma, large bowel leiomyoma, colorectalcancer, gall bladder cancer, and anal cancer.

In an embodiment, the gastrointestinal cancer is colorectal cancer.

In another embodiment, the cancer is a carcinoma. In yet anotherembodiment, the carcinoma is selected from pancreatic carcinoma,colorectal carcinoma, lung carcinoma, bladder carcinoma, gastriccarcinoma, esophageal carcinoma, breast carcinoma, head and neckcarcinoma, cervical skin carcinoma, and thyroid carcinoma.

In still another embodiment, the cancer is a hematopoietic malignancy.In an embodiment, the hematopoietic malignancy is selected from multiplemyeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.

In another embodiment, the cancer is a neoplasm. In yet anotherembodiment, the neoplasm is glioblastoma or sarcomas.

In certain embodiments, the disclosure provides a method for treating aKRAS-mediated disorder in a patient in need thereof, comprising the stepof administering to said patient a compound according to the invention,or a pharmaceutically acceptable composition thereof.

In some embodiments, diseases and indications that are treatable usingthe compounds of the present disclosure include, but are not limited tohematological cancers, sarcomas, lung cancers, gastrointestinal cancers,genitourinary tract cancers, liver cancers, bone cancers, nervous systemcancers, gynecological cancers, and skin cancers.

Exemplary hematological cancers include lymphomas and leukemias such asacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma(DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsedor refractory NHL and recurrent follicular), Hodgkin lymphoma,myeloproliferative diseases (e.g., primary myelofibrosis (PMF),polycythemia vera (PV), essential thrombocytosis (ET), 8p11myeloproliferative syndrome, myelodysplasia syndrome (MDS), T-cell acutelymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-celllymphoma, adult T-cell leukemia, Waldenstrom's Macroglubulinemia, hairycell lymphoma, marginal zone lymphoma, chronic myelogenic lymphoma andBurkitt's lymphoma.

Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma,osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma,myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma,lymphosarcoma, leiomyosarcoma, and teratoma.

Exemplary lung cancers include non-small cell lung cancer (NSCLC), smallcell lung cancer, bronchogenic carcinoma (squamous cell,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellularcarcinoma, bronchial adenoma and pleuropulmonary blastoma.

Exemplary gastrointestinal cancers include cancers of the esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (exocrinepancreatic carcinoma, ductal adenocarcinoma, insulinoma, glucagonoma,gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma,lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma,lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubularadenoma, villous adenoma, hamartoma, leiomyoma), colorectal cancer, gallbladder cancer and anal cancer.

Exemplary genitourinary tract cancers include cancers of the kidney(adenocarcinoma, Wilm's tumor [nephroblastoma], renal cell carcinoma),bladder and urethra (squamous cell carcinoma, transitional cellcarcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma) and urothelial carcinoma.

Exemplary liver cancers include hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, and hemangioma.

Exemplary bone cancers include, for example, osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant celltumors Exemplary nervous system cancers include cancers of the skull(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,meduoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma,glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,congenital tumors, neuro-ectodermal tumors), and spinal cord(neurofibroma, meningioma, glioma, sarcoma), neuroblastoma,Lhermitte-Duclos disease and pineal tumors.

Exemplary gynecological cancers include cancers of the breast (ductalcarcinoma, lobular carcinoma, breast sarcoma, triple-negative breastcancer, HER2-positive breast cancer, inflammatory breast cancer,papillary carcinoma), uterus (endometrial carcinoma), cervix (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma(serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), and fallopian tubes (carcinoma).

Exemplary skin cancers include melanoma, basal cell carcinoma, squamouscell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, molesdysplastic nevi, lipoma, angioma, dermatofibroma, and keloids.

Exemplary head and neck cancers include glioblastoma, melanoma,rhabdosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinomas,adenocarcinomas, oral cancer, laryngeal cancer, nasopharyngeal cancer,nasal and paranasal cancers, thyroid and parathyroid cancers, tumors ofthe eye, tumors of the lips and mouth and squamous head and neck cancer.

The compounds of the present disclosure can also be useful in theinhibition of tumor metastases.

In addition to oncogenic neoplasms, the compounds of the invention areuseful in the treatment of skeletal and chondrocyte disorders including,but not limited to, achrondroplasia, hypochondroplasia, dwarfism,thanatophoric dysplasia (TD) (clinical forms TD I and TD II), Apertsyndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevensoncutis gyrate syndrome, Pfeiffer syndrome, and craniosynostosissyndromes. In some embodiments, the present disclosure provides a methodfor treating a patient suffering from a skeletal and chondrocytedisorder.

In some embodiments, compounds described herein can be used to treatAlzheimer's disease, HIV, or tuberculosis.

As used herein, the term “8p11 myeloproliferative syndrome” is meant torefer to myeloid/lymphoid neoplasms associated with eosinophilia andabnormalities of FGFR1.

As used herein, the term “cell” is meant to refer to a cell that is invitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can bepart of a tissue sample excised from an organism such as a mammal. Insome embodiments, an in vitro cell can be a cell in a cell culture. Insome embodiments, an in vivo cell is a cell living in an organism suchas a mammal.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” KRAS with a compound described herein includes theadministration of a compound described herein to an individual orpatient, such as a human, having KRAS, as well as, for example,introducing a compound described herein into a sample containing acellular or purified preparation containing KRAS.

As used herein, the term “individual,” “subject,” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent such as an amountof any of the solid forms or salts thereof as disclosed herein thatelicits the biological or medicinal response in a tissue, system,animal, individual or human that is being sought by a researcher,veterinarian, medical doctor or other clinician. An appropriate“effective” amount in any individual case may be determined usingtechniques known to a person skilled in the art.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, immunogenicity or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase “pharmaceutically acceptable carrier orexcipient” refers to a pharmaceutically-acceptable material,composition, or vehicle, such as a liquid or solid filler, diluent,solvent, or encapsulating material. Excipients or carriers are generallysafe, non-toxic and neither biologically nor otherwise undesirable andinclude excipients or carriers that are acceptable for veterinary use aswell as human pharmaceutical use. In one embodiment, each component is“pharmaceutically acceptable” as defined herein. See, e.g., Remington:The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams &Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients,6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the AmericanPharmaceutical Association: 2009; Handbook of Pharmaceutical Additives,3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007;Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRCPress LLC: Boca Raton, Fla., 2009.

As used herein, the term “treating” or “treatment” refers to inhibitinga disease; for example, inhibiting a disease, condition, or disorder inan individual who is experiencing or displaying the pathology orsymptomology of the disease, condition, or disorder (i.e., arrestingfurther development of the pathology and/or symptomology) orameliorating the disease; for example, ameliorating a disease,condition, or disorder in an individual who is experiencing ordisplaying the pathology or symptomology of the disease, condition, ordisorder (i.e., reversing the pathology and/or symptomology) such asdecreasing the severity of the disease.

The term “prevent,” “preventing,” or “prevention” as used herein,comprises the prevention of at least one symptom associated with orcaused by the state, disease or disorder being prevented.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, can also beprovided in combination in a single embodiment (while the embodimentsare intended to be combined as if written in multiply dependent form).Conversely, various features of the invention which are, for brevity,described in the context of a single embodiment, can also be providedseparately or in any suitable subcombination.

Combination Therapies I. Cancer Therapies

Cancer cell growth and survival can be impacted by dysfunction inmultiple signaling pathways. Thus, it is useful to combine differentenzyme/protein/receptor inhibitors, exhibiting different preferences inthe targets which they modulate the activities of, to treat suchconditions. Targeting more than one signaling pathway (or more than onebiological molecule involved in a given signaling pathway) may reducethe likelihood of drug-resistance arising in a cell population, and/orreduce the toxicity of treatment.

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, immune-oncology agents, metabolic enzyme inhibitors,chemokine receptor inhibitors, and phosphatase inhibitors, as well astargeted therapies such as Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET,VEGFR, PDGFR, c-Kit, IGF-1R, RAF, FAK, and CDK4/6 kinase inhibitors suchas, for example, those described in WO 2006/056399 can be used incombination with the compounds of the present disclosure for treatmentof CDK2-associated diseases, disorders or conditions. Other agents suchas therapeutic antibodies can be used in combination with the compoundsof the present disclosure for treatment of CDK2-associated diseases,disorders or conditions. The one or more additional pharmaceuticalagents can be administered to a patient simultaneously or sequentially.

In some embodiments, the CDK2 inhibitor is administered or used incombination with a BCL2 inhibitor or a CDK4/6 inhibitor.

The compounds as disclosed herein can be used in combination with one ormore other enzyme/protein/receptor inhibitors therapies for thetreatment of diseases, such as cancer and other diseases or disordersdescribed herein. Examples of diseases and indications treatable withcombination therapies include those as described herein. Examples ofcancers include solid tumors and non-solid tumors, such as liquidtumors, blood cancers. Examples of infections include viral infections,bacterial infections, fungus infections or parasite infections. Forexample, the compounds of the present disclosure can be combined withone or more inhibitors of the following kinases for the treatment ofcancer: Akt1, Akt2, Akt3, BCL2, CDK4/6, TGF-PR, PKA, PKG, PKC,CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2,HER3, HER4, INS-R, IDH2, IGF-1R, IR-R, PDGFaR, PDGFPR, PI3K (alpha,beta, gamma, delta, and multiple or selective), CSF1R, KIT, FLK-II,KDR/FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, PARP, Ron,Sea, TRKA, TRKB, TRKC, TAM kinases (Axl, Mer, Tyro3), FLT3, VEGFR/Flt2,Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk,Fak, SYK, FRK, JAK, ABL, ALK and B-Raf. In some embodiments, thecompounds of the present disclosure can be combined with one or more ofthe following inhibitors for the treatment of cancer or infections.Non-limiting examples of inhibitors that can be combined with thecompounds of the present disclosure for treatment of cancer andinfections include an FGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4,e.g., pemigatinib (INCB54828), INCB62079), an EGFR inhibitor (also knownas ErB-1 or HER-1; e.g., erlotinib, gefitinib, vandetanib, orsimertinib,cetuximab, necitumumab, or panitumumab), a VEGFR inhibitor or pathwayblocker (e.g. bevacizumab, pazopanib, sunitinib, sorafenib, axitinib,regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab,lenvatinib, ziv-aflibercept), a PARP inhibitor (e.g., olaparib,rucaparib, veliparib or niraparib), a JAK inhibitor (JAK1 and/or JAK2;e.g., ruxolitinib or baricitinib; or JAK1; e.g., itacitinib (INCB39110),INCB052793, or INCB054707), an IDO inhibitor (e.g., epacadostat, NLG919,or BMS-986205, MK7162), an LSD1 inhibitor (e.g., GSK2979552, INCB59872and INCB60003), a TDO inhibitor, a PI3K-delta inhibitor (e.g.,parsaclisib (INCB50465) or INCB50797), a PI3K-gamma inhibitor such asPI3K-gamma selective inhibitor, a Pim inhibitor (e.g., INCB53914), aCSF1R inhibitor, a TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer;e.g., INCB081776), an adenosine receptor antagonist (e.g., A2a/A2breceptor antagonist), an HPK1 inhibitor, a chemokine receptor inhibitor(e.g., CCR2 or CCR5 inhibitor), a SHP1/2 phosphatase inhibitor, ahistone deacetylase inhibitor (HDAC) such as an HDAC8 inhibitor, anangiogenesis inhibitor, an interleukin receptor inhibitor, bromo andextra terminal family members inhibitors (for example, bromodomaininhibitors or BET inhibitors such as INCB54329 and INCB57643), c-METinhibitors (e.g., capmatinib), an anti-CD19 antibody (e.g.,tafasitamab), an ALK2 inhibitor (e.g., INCB00928); or combinationsthereof.

In some embodiments, the compound or salt described herein isadministered with a PI3Kδ inhibitor. In some embodiments, the compoundor salt described herein is administered with a JAK inhibitor. In someembodiments, the compound or salt described herein is administered witha JAK1 or JAK2 inhibitor (e.g., baricitinib or ruxolitinib). In someembodiments, the compound or salt described herein is administered witha JAK1 inhibitor. In some embodiments, the compound or salt describedherein is administered with a JAK1 inhibitor, which is selective overJAK2.

In addition, for treating cancer and other proliferative diseases,compounds described herein can be used in combination with targetedtherapies such as, e.g., c-MET inhibitors (e.g., capmatinib), ananti-CD19 antibody (e.g., tafasitamab), an ALK2 inhibitor (e.g.,INCB00928); or combinations thereof.

Example antibodies for use in combination therapy include, but are notlimited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g.,anti-VEGF-A), bevacizumab (AVASTIN™ e.g., anti-VEGF), panitumumab (e.g.,anti-EGFR), cetuximab (e.g., anti-EGFR), rituxan (e.g., anti-CD20), andantibodies directed to c-MET.

One or more of the following agents may be used in combination with thecompounds of the present disclosure and are presented as a non-limitinglist: a cytostatic agent, cisplatin, doxorubicin, taxotere, taxol,etoposide, irinotecan, camptosar, topotecan, paclitaxel, docetaxel,epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide,cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, IRESSA™(gefitinib), TARCEVA™ (erlotinib), antibodies to EGFR, intron, ara-C,adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine,ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, oxaliplatin, leucovirin, ELOXATIN™(oxaliplatin), pentostatine, vinblastine, vincristine, vindesine,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase,teniposide 17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone,Prednisone, Fluoxymesterone, Dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyltestosterone, prednisolone,triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, goserelin, carboplatin, hydroxyurea, amsacrine,procarbazine, mitotane, mitoxantrone, levamisole, navelbene,anastrazole, letrazole, capecitabine, reloxafine, droloxafine,hexamethylmelamine, avastin, HERCEPTIN™ (trastuzumab), BEXXAR™(tositumomab), VELCADE™ (bortezomib), ZEVALIN™ (ibritumomab tiuxetan),TRISENOX™ (arsenic trioxide), XELODA™ (capecitabine), vinorelbine,porfimer, ERBITUX™ (cetuximab), thiotepa, altretamine, melphalan,trastuzumab, lerozole, fulvestrant, exemestane, ifosfomide, rituximab,C225 (cetuximab), Campath (alemtuzumab), clofarabine, cladribine,aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine, Smi1,fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP, andMDL-101,731.

The compounds of the present disclosure can further be used incombination with other methods of treating cancers, for example bychemotherapy, irradiation therapy, tumor-targeted therapy, adjuvanttherapy, immunotherapy or surgery. Examples of immunotherapy includecytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), CRS-207immunotherapy, cancer vaccine, monoclonal antibody, bispecific ormulti-specific antibody, antibody drug conjugate, adoptive T celltransfer, Toll receptor agonists, RIG-1 agonists, oncolytic virotherapyand immunomodulating small molecules, including thalidomide or JAK1/2inhibitor, PI3Kδ inhibitor and the like. The compounds can beadministered in combination with one or more anti-cancer drugs, such asa chemotherapeutic agent. Examples of chemotherapeutics include any of:abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine,bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, busulfanintravenous, busulfan oral, calusterone, capecitabine, carboplatin,carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparinsodium, dasatinib, daunorubicin, decitabine, denileukin, denileukindiftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolonepropionate, eculizumab, epirubicin, erlotinib, estramustine, etoposidephosphate, etoposide, exemestane, fentanyl citrate, filgrastim,floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib,gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelinacetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinibmesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate,lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole,lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine,methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone,nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin,paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim,pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine,quinacrine, rasburicase, rituximab, ruxolitinib, sorafenib,streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide,teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan,toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard,valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, andzoledronate.

Additional examples of chemotherapeutics include proteasome inhibitors(e.g., bortezomib), thalidomide, revlimid, and DNA-damaging agents suchas melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include corticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include imatinib mesylate (GLEEVAC™),nilotinib, dasatinib, bosutinib, and ponatinib, and pharmaceuticallyacceptable salts. Other example suitable Bcr-Abl inhibitors include thecompounds, and pharmaceutically acceptable salts thereof, of the generaand species disclosed in U.S. Pat. No. 5,521,184, WO 04/005281, and U.S.Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include midostaurin, lestaurtinib,linifanib, sunitinib, sunitinib, maleate, sorafenib, quizartinib,crenolanib, pacritinib, tandutinib, PLX3397 and ASP2215, and theirpharmaceutically acceptable salts. Other example suitable Flt-3inhibitors include compounds, and their pharmaceutically acceptablesalts, as disclosed in WO 03/037347, WO 03/099771, and WO 04/046120.

Example suitable RAF inhibitors include dabrafenib, sorafenib, andvemurafenib, and their pharmaceutically acceptable salts. Other examplesuitable RAF inhibitors include compounds, and their pharmaceuticallyacceptable salts, as disclosed in WO 00/09495 and WO 05/028444.

Example suitable FAK inhibitors include VS-4718, VS-5095, VS-6062,VS-6063, B1853520, and GSK2256098, and their pharmaceutically acceptablesalts. Other example suitable FAK inhibitors include compounds, andtheir pharmaceutically acceptable salts, as disclosed in WO 04/080980,WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO01/014402.

Example suitable CDK4/6 inhibitors include palbociclib, ribociclib,trilaciclib, lerociclib, and abemaciclib, and their pharmaceuticallyacceptable salts. Other example suitable CDK4/6 inhibitors includecompounds, and their pharmaceutically acceptable salts, as disclosed inWO 09/085185, WO 12/129344, WO 11/101409, WO 03/062236, WO 10/075074,and WO 12/061156.

In some embodiments, the compounds of the disclosure can be used incombination with one or more other kinase inhibitors including imatinib,particularly for treating patients resistant to imatinib or other kinaseinhibitors.

In some embodiments, the compounds of the disclosure can be used incombination with a chemotherapeutic in the treatment of cancer, and mayimprove the treatment response as compared to the response to thechemotherapeutic agent alone, without exacerbation of its toxic effects.In some embodiments, the compounds of the disclosure can be used incombination with a chemotherapeutic provided herein. For example,additional pharmaceutical agents used in the treatment of multiplemyeloma, can include, without limitation, melphalan, melphalan plusprednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib).Further additional agents used in the treatment of multiple myelomainclude Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors. In someembodiments, the agent is an alkylating agent, a proteasome inhibitor, acorticosteroid, or an immunomodulatory agent. Examples of an alkylatingagent include cyclophosphamide (CY), melphalan (MEL), and bendamustine.In some embodiments, the proteasome inhibitor is carfilzomib. In someembodiments, the corticosteroid is dexamethasone (DEX). In someembodiments, the immunomodulatory agent is lenalidomide (LEN) orpomalidomide (POM). Additive or synergistic effects are desirableoutcomes of combining a CDK2 inhibitor of the present disclosure with anadditional agent.

The agents can be combined with the present compound in a single orcontinuous dosage form, or the agents can be administered simultaneouslyor sequentially as separate dosage forms.

The compounds of the present disclosure can be used in combination withone or more other inhibitors or one or more therapies for the treatmentof infections. Examples of infections include viral infections,bacterial infections, fungus infections or parasite infections.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with the compounds of thedisclosure where the dexamethasone is administered intermittently asopposed to continuously.

The compounds of Formula (I) or any of the formulas as described herein,a compound as recited in any of the claims and described herein, orsalts thereof can be combined with another immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides, and carbohydrate molecules), cells, and cellstransfected with genes encoding immune stimulating cytokines.Non-limiting examples of tumor vaccines that can be used includepeptides of melanoma antigens, such as peptides of gp100, MAGE antigens,Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to expressthe cytokine GM-CSF.

The compounds of Formula (I) or any of the formulas as described herein,a compound as recited in any of the claims and described herein, orsalts thereof can be used in combination with a vaccination protocol forthe treatment of cancer. In some embodiments, the tumor cells aretransduced to express GM-CSF. In some embodiments, tumor vaccinesinclude the proteins from viruses implicated in human cancers such asHuman Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) andKaposi's Herpes Sarcoma Virus (KHSV). In some embodiments, the compoundsof the present disclosure can be used in combination with tumor specificantigen such as heat shock proteins isolated from tumor tissue itself.In some embodiments, the compounds of Formula (I) or any of the formulasas described herein, a compound as recited in any of the claims anddescribed herein, or salts thereof can be combined with dendritic cellsimmunization to activate potent anti-tumor responses.

The compounds of the present disclosure can be used in combination withbispecific macrocyclic peptides that target Fe alpha or Fe gammareceptor-expressing effectors cells to tumor cells. The compounds of thepresent disclosure can also be combined with macrocyclic peptides thatactivate host immune responsiveness.

In some further embodiments, combinations of the compounds of thedisclosure with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant. The compounds of the present disclosure can be used incombination with bone marrow transplant for the treatment of a varietyof tumors of hematopoietic origin.

The compounds of Formula (I) or any of the formulas as described herein,a compound as recited in any of the claims and described herein, orsalts thereof can be used in combination with vaccines, to stimulate theimmune response to pathogens, toxins, and self-antigens. Examples ofpathogens for which this therapeutic approach may be particularlyuseful, include pathogens for which there is currently no effectivevaccine, or pathogens for which conventional vaccines are less thancompletely effective. These include, but are not limited to, HIV,Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania,Staphylococcus aureus, Pseudomonas Aeruginosa.

Viruses causing infections treatable by methods of the presentdisclosure include, but are not limit to human papillomavirus,influenza, hepatitis A, B, C or D viruses, adenovirus, poxvirus, herpessimplex viruses, human cytomegalovirus, severe acute respiratorysyndrome virus, Ebola virus, measles virus, herpes virus (e.g., VZV,HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), flaviviruses,echovirus, rhinovirus, coxsackie virus, cornovirus, respiratorysyncytial virus, mumps virus, rotavirus, measles virus, rubella virus,parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus,molluscum virus, poliovirus, rabies virus, JC virus and arboviralencephalitis virus.

Pathogenic bacteria causing infections treatable by methods of thedisclosure include, but are not limited to, Chlamydia, rickettsialbacteria, mycobacteria, staphylococci, streptococci, pneumococci,meningococci and conococci, Klebsiella, Proteus, Serratia, Pseudomonas,Legionella, diphtheria, Salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospirosis, and Lyme's disease bacteria.

Pathogenic fungi causing infections treatable by methods of thedisclosure include, but are not limited to, Candida (albicans, krusei,glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus(fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Pathogenic parasites causing infections treatable by methods of thedisclosure include, but are not limited to, Entamoeba histolytica,Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasiliensis.

When more than one pharmaceutical agent is administered to a patient,they can be administered simultaneously, separately, sequentially, or incombination (e.g., for more than two agents).

Methods for the safe and effective administration of most of thesechemotherapeutic agents are known to those skilled in the art. Inaddition, their administration is described in the standard literature.For example, the administration of many of the chemotherapeutic agentsis described in the “Physicians' Desk Reference” (PDR, e.g., 1996edition, Medical Economics Company, Montvale, N.J.), the disclosure ofwhich is incorporated herein by reference as if set forth in itsentirety.

II. Immune-checkpoint therapies Compounds of the present disclosure canbe used in combination with one or more immune checkpoint inhibitors forthe treatment of diseases, such as cancer or infections.

Exemplary immune checkpoint inhibitors include inhibitors against immunecheckpoint molecules such as CBL-B, CD20, CD28, CD40, CD70, CD122, CD96,CD73, CD47, CDK2, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM,arginase, HPK1, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4,BTLA, CTLA-4, LAG3, TIM3, TLR (TLR7/8), TIGIT, CD112R, VISTA, PD-1,PD-L1 and PD-L2. In some embodiments, the immune checkpoint molecule isa stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS,OX40, GITR and CD137. In some embodiments, the immune checkpointmolecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3,B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, TIGIT, and VISTA. Insome embodiments, the compounds provided herein can be used incombination with one or more agents selected from KIR inhibitors, TIGITinhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFRbeta inhibitors.

In some embodiments, the compounds provided herein can be used incombination with one or more agonists of immune checkpoint molecules,e.g., OX40, CD27, GITR, and CD137 (also known as 4-1BB).

In some embodiments, the inhibitor of an immune checkpoint molecule isanti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 or anti-PD-L1monoclonal antibody. In some embodiments, the anti-PD-1 or anti-PD-L1antibody is nivolumab, pembrolizumab, atezolizumab, durvalumab,avelumab, cemiplimab, atezolizumab, avelumab, tislelizumab,spartalizumab (PDR001), cetrelimab (JNJ-63723283), toripalimab (JS001),camrelizumab (SHR-1210), sintilimab (1B1308), AB122 (GLS-010), AMP-224,AMP-514/MEDI-0680, BMS936559, JTX-4014, BGB-108, SHR-1210, MEDI4736,FAZ053, BCD-100, KN035, CS1001, BAT1306, LZM009, AK105, HLX10, SHR-1316,CBT-502 (TQB2450), A167 (KL-A167), STI-A101 (ZKAB001), CK-301, BGB-A333,MSB-2311, HLX20, TSR-042, or LY3300054. In some embodiments, theinhibitor of PD-1 or PD-L1 is one disclosed in U.S. Pat. Nos. 7,488,802,7,943,743, 8,008,449, 8,168,757, 8,217, 149, or 10,308,644; U.S. Publ.Nos. 2017/0145025, 2017/0174671, 2017/0174679, 2017/0320875,2017/0342060, 2017/0362253, 2018/0016260, 2018/0057486, 2018/0177784,2018/0177870, 2018/0179179, 2018/0179201, 2018/0179202, 2018/0273519,2019/0040082, 2019/0062345, 2019/0071439, 2019/0127467, 2019/0144439,2019/0202824, 2019/0225601, 2019/0300524, or 2019/0345170; or PCT Pub.Nos. WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO2011066342, WO 2011159877, WO 2011082400, or WO 2011161699, which areeach incorporated herein by reference in their entirety. In someembodiments, the inhibitor of PD-L1 is INCB086550.

In some embodiments, the antibody is an anti-PD-1 antibody, e.g., ananti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab,camrelizumab, cetrelimab, toripalimab, sintilimab, AB122, AMP-224,JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, or TSR-042.In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab,cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, orsintilimab. In some embodiments, the anti-PD-1 antibody ispembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab.In some embodiments, the anti-PD-1 antibody is cemiplimab. In someembodiments, the anti-PD-1 antibody is spartalizumab. In someembodiments, the anti-PD-1 antibody is camrelizumab. In someembodiments, the anti-PD-1 antibody is cetrelimab. In some embodiments,the anti-PD-1 antibody is toripalimab. In some embodiments, theanti-PD-1 antibody is sintilimab. In some embodiments, the anti-PD-1antibody is AB122. In some embodiments, the anti-PD-1 antibody isAMP-224. In some embodiments, the anti-PD-1 antibody is JTX-4014. Insome embodiments, the anti-PD-1 antibody is BGB-108. In someembodiments, the anti-PD-1 antibody is BCD-100. In some embodiments, theanti-PD-1 antibody is BAT1306. In some embodiments, the anti-PD-1antibody is LZM009. In some embodiments, the anti-PD-1 antibody isAK105. In some embodiments, the anti-PD-1 antibody is HLX10. In someembodiments, the anti-PD-1 antibody is TSR-042. In some embodiments, theanti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In someembodiments, the anti-PD-1 monoclonal antibody is MGA012 (INCMGA0012;retifanlimab). In some embodiments, the anti-PD1 antibody is SHR-1210.Other anti-cancer agent(s) include antibody therapeutics such as 4-1BB(e.g., urelumab, utomilumab). In some embodiments, the inhibitor of animmune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonalantibody is atezolizumab, avelumab, durvalumab, tislelizumab,BMS-935559, MEDI4736, atezolizumab (MPDL3280A; also known as RG7446),avelumab (MSB0010718C), FAZ053, KN035, CS1001, SHR-1316, CBT-502, A167,STI-A101, CK-301, BGB-A333, MSB-2311, HLX20, or LY3300054. In someembodiments, the anti-PD-L1 antibody is atezolizumab, avelumab,durvalumab, or tislelizumab. In some embodiments, the anti-PD-L1antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibodyis avelumab. In some embodiments, the anti-PD-L1 antibody is durvalumab.In some embodiments, the anti-PD-L1 antibody is tislelizumab. In someembodiments, the anti-PD-L1 antibody is BMS-935559. In some embodiments,the anti-PD-L1 antibody is MEDI4736. In some embodiments, the anti-PD-L1antibody is FAZ053. In some embodiments, the anti-PD-L1 antibody isKN035. In some embodiments, the anti-PD-L1 antibody is CS1001. In someembodiments, the anti-PD-L1 antibody is SHR-1316. In some embodiments,the anti-PD-L1 antibody is CBT-502. In some embodiments, the anti-PD-L1antibody is A167. In some embodiments, the anti-PD-L1 antibody isSTI-A101. In some embodiments, the anti-PD-L1 antibody is CK-301. Insome embodiments, the anti-PD-L1 antibody is BGB-A333. In someembodiments, the anti-PD-L1 antibody is MSB-2311. In some embodiments,the anti-PD-L1 antibody is HLX20. In some embodiments, the anti-PD-L1antibody is LY3300054.

In some embodiments, the inhibitor of an immune checkpoint molecule is asmall molecule that binds to PD-L1, or a pharmaceutically acceptablesalt thereof. In some embodiments, the inhibitor of an immune checkpointmolecule is a small molecule that binds to and internalizes PD-L1, or apharmaceutically acceptable salt thereof. In some embodiments, theinhibitor of an immune checkpoint molecule is a compound selected fromthose in US 2018/0179201, US 2018/0179197, US 2018/0179179, US2018/0179202, US 2018/0177784, US 2018/0177870, U.S. Ser. No. 16/369,654(filed Mar. 29, 2019), and U.S. Ser. No. 62/688,164, or apharmaceutically acceptable salt thereof, each of which is incorporatedherein by reference in its entirety.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of KIR, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

In some embodiments, the inhibitor is MCLA-145.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In someembodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab,AGEN1884, or CP-675,206.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of LAG3, e.g., an anti-LAG3 antibody. In some embodiments,the anti-LAG3 antibody is BMS-986016, LAG525, INCAGN2385, or eftilagimodalpha (IMP321).

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CD73. In some embodiments, the inhibitor of CD73 isoleclumab.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of TIGIT. In some embodiments, the inhibitor of TIGIT isOMP-31M32.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of VISTA. In some embodiments, the inhibitor of VISTA isJNJ-61610588 or CA-170.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of B7-H3. In some embodiments, the inhibitor of B7-H3 isenoblituzumab, MGD009, or 8H9.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of KIR. In some embodiments, the inhibitor of KIR islirilumab or IPH4102.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of A2aR. In some embodiments, the inhibitor of A2aR isCPI-444.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of TGF-beta. In some embodiments, the inhibitor of TGF-betais trabedersen, galusertinib, or M7824.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of PI3K-gamma. In some embodiments, the inhibitor ofPI3K-gamma is IPI-549.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CD47. In some embodiments, the inhibitor of CD47 isHu5F9-G4 or TTI-621.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CD73. In some embodiments, the inhibitor of CD73 isMEDI9447.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CD70. In some embodiments, the inhibitor of CD70 iscusatuzumab or BMS-936561.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of TIM3, e.g., an anti-TIM3 antibody. In some embodiments,the anti-TIM3 antibody is INCAGN2390, MBG453, or TSR-022.

In some embodiments, the inhibitor of an immune checkpoint molecule isan inhibitor of CD20, e.g., an anti-CD20 antibody. In some embodiments,the anti-CD20 antibody is obinutuzumab or rituximab.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of OX40, CD27, CD28, GITR, ICOS, CD40, TLR7/8, and CD137 (alsoknown as 4-1BB).

In some embodiments, the agonist of CD137 is urelumab. In someembodiments, the agonist of CD137 is utomilumab.

In some embodiments, the agonist of an immune checkpoint molecule is aninhibitor of GITR. In some embodiments, the agonist of GITR is TRX518,MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, MED11873, orMEDI6469. In some embodiments, the agonist of an immune checkpointmolecule is an agonist of OX40, e.g., OX40 agonist antibody or OX40Lfusion protein. In some embodiments, the anti-OX40 antibody isINCAGN01949, MED10562 (tavolimab), MOXR-0916, PF-04518600, GSK3174998,BMS-986178, or 9B12. In some embodiments, the OX40L fusion protein isMEDI6383.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of CD40. In some embodiments, the agonist of CD40 is CP-870893,ADC-1013, CDX-1140, SEA-CD40, R07009789, JNJ-64457107, APX-005M, or ChiLob 7/4.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of ICOS. In some embodiments, the agonist of ICOS isGSK-3359609, JTX-2011, or MEDI-570.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of CD28. In some embodiments, the agonist of CD28 istheralizumab.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of CD27. In some embodiments, the agonist of CD27 is varlilumab.

In some embodiments, the agonist of an immune checkpoint molecule is anagonist of TLR7/8. In some embodiments, the agonist of TLR7/8 isMEDI9197.

The compounds of the present disclosure can be used in combination withbispecific antibodies. In some embodiments, one of the domains of thebispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3,CD137, ICOS, CD3 or TGFβ receptor. In some embodiments, the bispecificantibody binds to PD-1 and PD-L1. In some embodiments, the bispecificantibody that binds to PD-1 and PD-L1 is MCLA-136. In some embodiments,the bispecific antibody binds to PD-L1 and CTLA-4. In some embodiments,the bispecific antibody that binds to PD-L1 and CTLA-4 is AK104.

In some embodiments, the compounds of the disclosure can be used incombination with one or more metabolic enzyme inhibitors. In someembodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1,TDO, or arginase. Examples of IDO1 inhibitors include epacadostat,NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099 and LY338196.Inhibitors of arginase inhibitors include INCB1158.

As provided throughout, the additional compounds, inhibitors, agents,etc. can be combined with the present compound in a single or continuousdosage form, or they can be administered simultaneously or sequentiallyas separate dosage forms.

Formulation, Dosage Forms and Administration

When employed as pharmaceuticals, the compounds of the presentdisclosure can be administered in the form of pharmaceuticalcompositions. Thus, the present disclosure provides a compositioncomprising a compound of Formula I, II, or any of the formulas asdescribed herein, a compound as recited in any of the claims anddescribed herein, or a pharmaceutically acceptable salt thereof, or anyof the embodiments thereof, and at least one pharmaceutically acceptablecarrier or excipient. These compositions can be prepared in a mannerwell known in the pharmaceutical art, and can be administered by avariety of routes, depending upon whether local or systemic treatment isindicated and upon the area to be treated. Administration may be topical(including transdermal, epidermal, ophthalmic and to mucous membranesincluding intranasal, vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be,e.g., by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the compound of the present disclosure or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers or excipients. In someembodiments, the composition is suitable for topical administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, e.g., a capsule, sachet, paper, orother container. When the excipient serves as a diluent, it can be asolid, semi-solid, or liquid material, which acts as a vehicle, carrieror medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, e.g., up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g., about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art see, e.g., WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

In some embodiments, the pharmaceutical composition comprises silicifiedmicrocrystalline cellulose (SMCC) and at least one compound describedherein, or a pharmaceutically acceptable salt thereof. In someembodiments, the silicified microcrystalline cellulose comprises about98% microcrystalline cellulose and about 2% silicon dioxide w/w.

In some embodiments, the composition is a sustained release compositioncomprising at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient. In some embodiments, the composition comprises atleast one compound described herein, or a pharmaceutically acceptablesalt thereof, and at least one component selected from microcrystallinecellulose, lactose monohydrate, hydroxypropyl methylcellulose andpolyethylene oxide. In some embodiments, the composition comprises atleast one compound described herein, or a pharmaceutically acceptablesalt thereof, and microcrystalline cellulose, lactose monohydrate andhydroxypropyl methylcellulose. In some embodiments, the compositioncomprises at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and microcrystalline cellulose, lactosemonohydrate and polyethylene oxide. In some embodiments, the compositionfurther comprises magnesium stearate or silicon dioxide. In someembodiments, the microcrystalline cellulose is Avicel PH102™. In someembodiments, the lactose monohydrate is Fast-flo 316™. In someembodiments, the hydroxypropyl methylcellulose is hydroxypropylmethylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/orhydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel K00LV™). Insome embodiments, the polyethylene oxide is polyethylene oxide WSR 1105(e.g., Polyox WSR 1105™).

In some embodiments, a wet granulation process is used to produce thecomposition. In some embodiments, a dry granulation process is used toproduce the composition.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1,000 mg (1 g), more usually about 100mg to about 500 mg, of the active ingredient. In some embodiments, eachdosage contains about 10 mg of the active ingredient. In someembodiments, each dosage contains about 50 mg of the active ingredient.In some embodiments, each dosage contains about 25 mg of the activeingredient. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient.

The components used to formulate the pharmaceutical compositions are ofhigh purity and are substantially free of potentially harmfulcontaminants (e.g., at least National Food grade, generally at leastanalytical grade, and more typically at least pharmaceutical grade).Particularly for human consumption, the composition is preferablymanufactured or formulated under Good Manufacturing Practice standardsas defined in the applicable regulations of the U.S. Food and DrugAdministration. For example, suitable formulations may be sterile and/orsubstantially isotonic and/or in full compliance with all GoodManufacturing Practice regulations of the U.S. Food and DrugAdministration.

The active compound may be effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms and the like.

The therapeutic dosage of a compound of the present invention can varyaccording to, e.g., the particular use for which the treatment is made,the manner of administration of the compound, the health and conditionof the patient, and the judgment of the prescribing physician. Theproportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 □g/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, formulation of the excipient, and its route of administration.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, e.g., about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, e.g., liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.,glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, e.g., glycerol,hydroxyethyl cellulose, and the like. In some embodiments, topicalformulations contain at least about 0.1, at least about 0.25, at leastabout 0.5, at least about 1, at least about 2 or at least about 5 wt %of the compound of the invention. The topical formulations can besuitably packaged in tubes of, e.g., 100 g which are optionallyassociated with instructions for the treatment of the select indication,e.g., psoriasis or other skin condition.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers or stabilizers will resultin the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention can varyaccording to, e.g., the particular use for which the treatment is made,the manner of administration of the compound, the health and conditionof the patient, and the judgment of the prescribing physician. Theproportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, formulation of the excipient, and its route of administration.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled compounds ofthe disclosure (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in imaging techniques but also in assays, both in vitroand in vivo, for localizing and quantitating KRAS protein in tissuesamples, including human, and for identifying KRAS ligands by inhibitionbinding of a labeled compound. Substitution of one or more of the atomsof the compounds of the present disclosure can also be useful ingenerating differentiated ADME (Adsorption, Distribution, Metabolism andExcretion). Accordingly, the present invention includes KRAS bindingassays that contain such labeled or substituted compounds.

The present disclosure further includes isotopically-labeled compoundsof the disclosure. An “isotopically” or “radio-labeled” compound is acompound of the disclosure where one or more atoms are replaced orsubstituted by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number typically found in nature (i.e.,naturally occurring). Suitable radionuclides that may be incorporated incompounds of the present disclosure include but are not limited to ²H(also written as D for deuterium), ³H (also written as T for tritium),¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br,⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. For example, one or more hydrogenatoms in a compound of the present disclosure can be replaced bydeuterium atoms (e.g., one or more hydrogen atoms of a C₁₋₆ alkyl groupof Formula I, II, or any formulae provided herein can be optionallysubstituted with deuterium atoms, such as —CD₃ being substituted for—CH₃). In some embodiments, alkyl groups in Formula I, II, or anyformulae provided herein can be perdeuterated.

One or more constituent atoms of the compounds presented herein can bereplaced or substituted with isotopes of the atoms in natural ornon-natural abundance. In some embodiments, the compound includes atleast one deuterium atom. In some embodiments, the compound includes twoor more deuterium atoms. In some embodiments, the compound includes 1-2,1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of thehydrogen atoms in a compound can be replaced or substituted by deuteriumatoms.

Synthetic methods for including isotopes into organic compounds areknown in the art (Deuterium Labeling in Organic Chemistry by Alan F.Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissanceof H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and JochenZimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistryof Isotopic Labelling by James R. Hanson, Royal Society of Chemistry,2011). Isotopically labeled compounds can be used in various studiessuch as NMR spectroscopy, metabolism experiments, and/or assays.

Substitution with heavier isotopes, such as deuterium, may affordcertain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances. (seee.g., A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al.J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular,substitution at one or more metabolism sites may afford one or more ofthe therapeutic advantages.

The radionuclide that is incorporated in the instant radio-labeledcompounds will depend on the specific application of that radio-labeledcompound. For example, for in vitro adenosine receptor labeling andcompetition assays, compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹Ior ³⁵S can be useful. For radio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I,¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br can be useful.

It is understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments, the radionuclide is selected from ³H, ¹⁴C, ¹²⁵I, ³⁵S and⁸²Br.

The present disclosure can further include synthetic methods forincorporating radio-isotopes into compounds of the disclosure. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and an ordinary skill in the art will readilyrecognize the methods applicable for the compounds of disclosure.

A labeled compound of the invention can be used in a screening assay toidentify and/or evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a KRAS protein by monitoring itsconcentration variation when contacting with the KRAS, through trackingof the labeling. For example, a test compound (labeled) can be evaluatedfor its ability to reduce binding of another compound which is known tobind to a KRAS protein (i.e., standard compound). Accordingly, theability of a test compound to compete with the standard compound forbinding to the KRAS protein directly correlates to its binding affinity.Conversely, in some other screening assays, the standard compound islabeled and test compounds are unlabeled. Accordingly, the concentrationof the labeled standard compound is monitored in order to evaluate thecompetition between the standard compound and the test compound, and therelative binding affinity of the test compound is thus ascertained.

Kits

The present disclosure also includes pharmaceutical kits useful, e.g.,in the treatment or prevention of diseases or disorders associated withthe activity of KRAS, such as cancer or infections, which include one ormore containers containing a pharmaceutical composition comprising atherapeutically effective amount of a compound of Formula I, II, or anyof the embodiments thereof. Such kits can further include one or more ofvarious conventional pharmaceutical kit components, such as, e.g.,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results.

The compounds of the Examples have been found to inhibit the activity ofKRAS according to at least one assay described herein.

EXAMPLES

Experimental procedures for compounds of the invention are providedbelow. Preparatory LC-MS purifications of some of the compounds preparedwere performed on Waters mass directed fractionation systems. The basicequipment setup, protocols, and control software for the operation ofthese systems have been described in detail in the literature. See e.g.“Two-Pump At Column Dilution Configuration for Preparative LC-MS”, K.Blom, J. Combi. Chem., 4, 295 (2002); “Optimizing Preparative LC-MSConfigurations and Methods for Parallel Synthesis Purification”, K.Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi.Chem., 5, 670 (2003); and “Preparative LC-MS Purification: ImprovedCompound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A.Combs, J. Combi. Chem., 6, 874-883 (2004). The compounds separated weretypically subjected to analytical liquid chromatography massspectrometry (LCMS) for purity check.

The compounds separated were typically subjected to analytical liquidchromatography mass spectrometry (LCMS) for purity check under thefollowing conditions: Instrument; Agilent 1100 series, LC/MSD, Column:Waters Sunfire™ C₁₈ 5 μm particle size, 2.1×5.0 mm, Buffers: mobilephase A: 0.025% TFA in water and mobile phase B: acetonitrile; gradient2% to 80% of B in 3 minutes with flow rate 2.0 mL/minute.

Some of the compounds prepared were also separated on a preparativescale by reverse-phase high performance liquid chromatography (RP-HPLC)with MS detector or flash chromatography (silica gel) as indicated inthe Examples. Typical preparative reverse-phase high performance liquidchromatography (RP-HPLC) column conditions are as follows:

pH=2 purifications: Waters Sunfire™ C₁₈ 5 μm particle size, 19×100 mmcolumn, eluting with mobile phase A: 0.1% TFA (trifluoroacetic acid) inwater and mobile phase B: acetonitrile; the flow rate was 30 mL/minute,the separating gradient was optimized for each compound using theCompound Specific Method Optimization protocol as described in theliterature [see “Preparative LCMS Purification: Improved CompoundSpecific Method Optimization”, K. Blom, B. Glass, R. Sparks, A. Combs,J. Comb. Chem., 6, 874-883 (2004)].

Typically, the flow rate used with the 30×100 mm column was 60mL/minute. pH=10 purifications: Waters XBridge C₁₈ 5 μm particle size,19×100 mm column, eluting with mobile phase A: 0.15% NH₄OH in water andmobile phase B: acetonitrile; the flow rate was 30 mL/minute, theseparating gradient was optimized for each compound using the CompoundSpecific Method Optimization protocol as described in the literature[See “Preparative LCMS Purification: Improved Compound Specific MethodOptimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem.,6, 874-883 (2004)]. Typically, the flow rate used with 30×100 mm columnwas 60 mL/minute.”

The following abbreviations may be used herein: AcOH (acetic acid); Ac₂O(acetic anhydride); aq. (aqueous); atm. (atmosphere(s)); Boc(t-butoxycarbonyl); BOP((benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate); br (broad); Cbz (carboxybenzyl); calc.(calculated); d (doublet); dd (doublet of doublets); DBU(1,8-diazabicyclo[5.4.0]undec-7-ene); DCM (dichloromethane); DIAD (N,N′-diisopropyl azidodicarboxylate); DIEA (N,N-diisopropylethylamine);DIPEA (N, N-diisopropylethylamine); DIBAL (diisobutylaluminium hydride);DMF (N, N-dimethylformamide); Et (ethyl); EtOAc (ethyl acetate); FCC(flash column chromatography); g (gram(s)); h (hour(s)); HATU (N, N, N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate);HCl (hydrochloric acid); HPLC (high performance liquid chromatography);Hz (hertz); J (coupling constant); LCMS (liquid chromatography-massspectrometry); LDA (lithium diisopropylamide); m (multiplet); M (molar);mCPBA (3-chloroperoxybenzoic acid); MS (Mass spectrometry); Me (methyl);MeCN (acetonitrile); MeOH (methanol); mg (milligram(s)); min.(minutes(s)); mL (milliliter(s)); mmol (millimole(s)); N (normal); NCS(N-chlorosuccinimide); NEt₃ (triethylamine); nM (nanomolar); NMP(N-methylpyrrolidinone); NMR (nuclear magnetic resonance spectroscopy);OTf (trifluoromethanesulfonate); Ph (phenyl); pM (picomolar); PPT(precipitate); RP-HPLC (reverse phase high performance liquidchromatography); r.t. (room temperature), s (singlet); t (triplet ortertiary); TBS (tert-butyldimethylsilyl); tert (tertiary); tt (tripletof triplets); TFA (trifluoroacetic acid); THF (tetrahydrofuran); μg(microgram(s)); μL (microliter(s)); μM (micromolar); wt % (weightpercent). Brine is saturated aqueous sodium chloride. In vacuo is undervacuum.

Intermediate 1. 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline

Step 1. 2-amino-4-bromo-3-fluoro-5-iodobenzoic acid

NIS (9.61 g, 42.7 mmol) was added to a solution of2-amino-4-bromo-3-fluorobenzoic acid (10.0 g, 42.7 mmol) in DMF (100 mL)and then the reaction was stirred at 80° C. for 6 h. The mixture wascooled with ice water and then water (150 mL) was added and stirred for20 min, the precipitate was filtered and washed with water, dried toprovide the desired product as a solid. LCMS calculated for C₇H₅BrFINO₂(M+H)⁺: m/z=359.9; found 359.8.

Step 2. 7-bromo-8-fluoro-6-iodo-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

To a solution of 2-amino-4-bromo-3-fluoro-5-iodobenzoic acid (8.4 g,23.34 mmol) in 1,4-Dioxane (200 mL) was added triphosgene (6.34 g, 21.37mmol), and stirred at 100° C. for 1 h. After cooling to r.t., ice wasadded until a solid precipitated. The mixture was then fully dilutedwith water (final volume˜400 mL) and the solid collected by filtrationthen air dried. The crude product was used in the next step withoutfurther purification.

Step 3. 7-bromo-8-fluoro-6-iodo-3-nitroquinoline-2,4-diol

DIPEA (6.06 ml, 34.7 mmol) was added to a solution of ethyl2-nitroacetate (4.62 g, 17.36 mmol) in toluene (10.0 mL) at r.t. andstirred for 10 min.7-Bromo-8-fluoro-6-iodo-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (6.7 g,17.36 mmol) was then added to the reaction mixture and the reaction wasstirred at 95° C. for 3 h. The reaction was cooled with ice water andthen 1 N HCl (40 mL) was added. The solid precipitate was collected viafiltration then washed with small amount of ethyl acetate to provide thedesired product as a yellow solid (6 g, 81%). LCMS calculated forC₉H₄BrFIN₂O₄ (M+H)⁺: m/z=428.8; found 428.8.

Step 4. 7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline

DIPEA (3.67 mL, 21.03 mmol) was added to a mixture of7-bromo-8-fluoro-6-iodo-3-nitroquinoline-2,4-diol (4.51 g, 10.51 mmol)in POCl₃ (4.9 mL, 52.6 mmol) and then the reaction was stirred at 105°C. for 3 h. The solvent was removed under vacuum and then azeotropedwith toluene 3 times to provide the crude material which was used in thenext step without further purification.

Intermediate 2. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

Step 1. tert-butyl(2S,4S)-4-((3-amino-7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodoquinolin-4-yl)amino)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

DIPEA (15.37 ml, 88 mmol) was added to a solution of7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1)(20.49 g, 44 mmol) and tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate(Intermediate 6) (13.83 g, 44 mmol) in CH₂Cl₂ (100 mL) at roomtemperature. The reaction was stirred at 50° C. for 3 h. Upon fullconversion, N,N,3-trimethylazetidin-3-amine dihydrochloride (9.98 g,52.8 mmol) and another 2 equiv of DIPEA (15.37 mL, 88 mmol) were addedto the reaction mixture and stirred at 50° C. overnight. The reactioncontent was transferred to a separatory funnel, and washed withsaturated NH₄Cl (200 mL) and brine (100 mL). The organic phase was driedover MgSO₄ and concentrated.

The concentrated residue was redissolved in MeOH (50 mL), CH₂Cl₂ (10 mL)and aqueous ammonium hydroxide (57 mL, 440 mmol). Sodium dithionite (23g, 132 mmol) was added in one portion then the reaction was stirredvigorously at room temperature overnight. Upon completion, the reactionwas quenched with the addition of H₂O (100 mL), and extracted withCH₂Cl₂ (100 mL). The organic phase was washed with H₂O twice, dried overNa₂SO₄, then concentrated to afford the desired diamine product as a redviscous oil (18.21 g, 56% over 2 steps). LCMS calculated forC₃₁H₄₆BrFIN₆O₄ (M+H)⁺: m/z=791.2; found: 791.1.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

The crude diamine from step 1 (18.21 g, 23 mmol) was dissolved inglacial acetic acid (57.9 mL, 1012 mmol). Sodium nitrite (2.38 g, 34.5mmol) was added in one portion. The reaction was stirred at roomtemperature for 2 h. Upon completion, the reaction contents were pouredinto vigorously stirred ice water. The precipitated solids werecollected via filtration, washed with NaHCO₃, water, and diethyl ether.The solids were then dried under vacuum to provide the desired productas a brown solid (15 g, 81% yield). LCMS calculated for C₃₁H₄₃BrFIN₇O₄(M+H)⁺: m/z=802.2; found 802.1.

Intermediate 3. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

Step 1. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

A pressure vessel was charged with Intermediate 2 (1.10 g, 1.37 mmol),copper(I) iodide (39 mg, 0.21 mmol), 1,10-phenanthroline (37 mg, 0.21mmol) and potassium fluoride (239 mg, 411 mmol). DMSO (2.74 mL) wasadded, and the vessel is flushed with N₂ for 5 min. Trimethyl borate(0.46 ml, 4.11 mmol) and trimethyl(trifluoromethyl)silane (0.61 mL, 4.11mmol) were added before the pressure vessel was sealed and heated to 80°C. overnight. The vessel was allowed to cool to room temperature, thencooled in an ice batch and opened carefully. The reaction mixture wasdiluted with EtOAc (20 mL) and washed with NaHCO₃, brine, andconcentrated. The crude product was purified on silica gel (20 g,50-100% EtOAc in CH₂Cl₂) to yield a brown solid (634 mg, 62% yield).LCMS calculated for C₃₂H₄₃BrF₄N₇O₄ (M+H)⁺: m/z=744.3; found: 744.2.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate(6.5 g, 8.7 mmol) was dissolved in dioxane (20 mL) and lithium hydroxidesolution (7.27 mL, 6 M in H₂O) was added. The reaction was heated to 80°C. overnight. Upon full hydrolysis of the tert-butyl ester, saturatedNH₄Cl (100 mL) was added, and the reaction was extracted with EtOAc(3×100 mL). The combined organic layers were dried over Na₂SO₄ andconcentrated to dryness to afford2-((2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(tert-butoxycarbonyl)piperidin-2-yl)aceticacid. LCMS calculated for C₂₈H₃₅BrF₄N₇O₄ (M+H)⁺: m/z=688.2; found 688.1.

The carboxylic acid was re-dissolved in THF (20 mL). DIPEA (3.1 mL, 17.5mmol) was added and the reaction mixture was cooled to 0° C. Isobutylchloroformate (1.7 mL, 13.1 mmol) was then added. After stirring at 0°C. for 20 min, ammonium hydroxide (11.3 mL, 87 mmol) was added and themixture was stirred for an additional 10 min. Upon completion, thereaction was diluted with EtOAc (20 mL), washed with brine, dried overMgSO₄, concentrated to afford tert-butyl(2S,4S)-2-(2-amino-2-oxoethyl)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₂₈H₃₆BrF₄N₈O₃ (M+H)⁺: m/z=687.2; found 687.4.

The crude amide was re-dissolved in THF (20 mL) and cooled to 0° C.Triethylamine (4.9 mL, 34.9 mmol) and TFAA (1.8 mL, 13.1 mol) were addedsequentially. After stirring for 1 hour, the reaction was quenched withthe addition of aq. NaHCO₃ (50 mL), extracted with EtOAc (50 mL), driedover Na₂SO₄, concentrated, and purified on silica (100 g, 0-100% EtOAcin CH₂Cl₂) to yield the title product as a yellow solid (4.3 g, 74%yield over 3 steps). LCMS calculated for C₂₈H₃₄BrF₄N₈O₂ (M+H)⁺:m/z=669.2; found 669.4.

Intermediate 4. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

Step 1. tert-butyl(2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

DIPEA (1.92 ml, 11 mmol) was added to a solution of7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1)(2.56 g, 5.5 mmol) and tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate(Intermediate 6) (1.73 g, 5.5 mmol) in CH₂Cl₂ (10 mL) at roomtemperature. The reaction was stirred at 50° C. for 3 h. Upon fullconversion, The reaction content was transferred to a separatory funnel,and washed with saturated NH₄Cl (50 mL) and brine (50 mL). The organicphase was dried over MgSO₄ and concentrated.

The concentrated residue was redissolved in MeOH (5 mL), CH₂Cl₂ (5 mL)and aqueous ammonium hydroxide (7.3 mL, 55 mmol). Sodium dithionite(2.88 g, 16.5 mmol) was added in one portion then the reaction wasstirred vigorously at room temperature overnight. Upon completion, thereaction was quenched with the addition of H₂O (10 mL), and extractedwith CH₂Cl₂ (50 mL). The organic phase was washed with H₂O twice, driedover Na₂SO₄, then concentrated to afford the desired diamine product.

The crude diamine was dissolved in glacial acetic acid (7.0 mL). Sodiumnitrite (0.76 g, 11 mmol) was added in one portion. The reaction wasstirred at room temperature for 20 mins. Upon completion, the reactioncontents were poured into vigorously stirred ice water. The reactionmixture was extracted with DCM. The crude product was purified on silicagel (50 g, 0-100% EtOAc in CH₂Cl₂) to provide the desired product (1.8g, 70% yield). LCMS calculated for C₂₅H₃₀BrClFIN₅O₄ (M+H)⁺: m/z=724.0;found: 724.0.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

tert-butyl(2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate(1.44 g, 1.99 mmol) was dissolved in CH₂Cl₂ (5 mL) and MeOH (5 mL) andstirred at room temperature until homogeneous. Sodium thiomethoxide(0.28 g, 3.97 mmol) was added in one portion. After stirring for 1 h,the reaction was quenched by saturated NH₄Cl (10 mL) and extracted withEtOAc (20 mL), then washed with NaHCO₃. The combined organic layers weredried over MgSO4, filtered, and concentrated. LCMS calculated forC₂₆H₃₃BrFIN₅O₄S (M+H)⁺: m/z=736.1; found: 736.0.

Intermediate 5. tert-butyl4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 4, replacing tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylatewith tert-butyl 4-aminopiperidine-1-carboxylate. LCMS calculated forC₂₀H₂₃BrFIN₅O₂S (M+H)⁺: m/z=622.0; found: 622.1.

Intermediate 6. tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

Step 1. tert-butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate

A solution of 2.0 M LDA (100 mL, 200 mmol) in anhydrous THF (223 mL) wascooled to −78° C. for 1 h, and then tert-butyl acetate (26.9 mL, 200mmol) was added dropwise with stirring over 20 min. After an additional40 minutes maintained at −78° C., a solution of ethyl(R)-4-cyano-3-hydroxybutanoate (10.5 g, 66.8 mmol) was added dropwise.The mixture was allowed to stir at −40° C. for 4 h, and then anappropriate amount of HCl (2 M) was added to the mixture, keeping pH ˜6.During this quench, the temperature of the mixture was maintained at−10° C. Upon completion, the temperature of the mixture was cooled to 0°C. The mixture was extracted with ethyl acetate (3×100 mL). The combinedorganic layer was washed with NaHCO₃ (100 mL) and brine (100 mL), driedover anhydrous Na₂SO₄, and concentrated to provide the material asyellow oil (15.0 g, 99% yield).

Step 2. tert-butyl(2S,4R)-2-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxypiperidine-1-carboxylate

A solution of tert-butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate (15.0 g,66.0 mmol) in acetic acid (110 ml) was treated with platinum (IV) oxidehydrate (0.868 g, 3.30 mmol). The Parr bottle was evacuated andbackfilled with H₂ three times and stirred under a H₂ atmosphere (45psi, recharged 4 times) at 22° C. for 3 h. The mixture was filteredthrough Celite and the filter cake was washed with EtOH. The filtratewas concentrated to yield product with a ˜9:1 cis:trans diastereomerratio.

The residue was dissolved in methanol (100 mL) then Boc-anhydride (15.32ml, 66.0 mmol), sodium carbonate (13.99 g, 132 mmol) was added. Thereaction mixture was stirred at room temperature overnight. The mixturewas filtered and concentrated. The residue was purified with silica gelcolumn to give the desired product (11.7 g, 56%). LCMS calculated forC₁₆H₂₉NNaO₅ (M+Na)⁺: m/z=338.2; found: 338.2.

Step 3. tert-butyl(2S,4S)-4-azido-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

To a solution of tert-butyl(2S,4R)-2-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxypiperidine-1-carboxylate(2.10 g, 6.66 mmol) in DCM (33 ml) at 0° C. was added TEA (1.58 ml,11.32 mmol) and Ms-Cl (0.67 mL, 8.66 mmol). After stirring for 1 h, Thereaction was diluted with water and organic layer was separated anddried over Na₂SO₄, filtered and concentrated. The resulting residue wasdissolved in DMF and sodium azide (1.3 g, 20 mmol) was added and thereaction mixture was heated at 70° C. for 5 h. After cooling to rt, thereaction was diluted with EtOAc and water. The organic layer wasseparated and dried over Na₂SO₄, filtered and concentrated. The residuewas purified with silica gel column to give the desired product (1.90 g,84%). LCMS calculated for (Product-Boc) C₁₁H₂₁N₄O₂ (M+H)⁺: m/z=241.2;found: 241.2.

Step 4. tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

To a solution of tert-butyl(2S,4S)-4-azido-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate(1.9 g, 5.58 mmol) in methanol (27.9 ml) was added 10% palladium oncarbon (0.594 g, 0.558 mmol). The reaction mixture was evacuated undervacuum and refilled with H₂, stirred at rt for 2 h. The reaction mixturewas filtered through a pad of Celite and washed with methanol. Thefiltrate was concentrated and used as is (1.5 g, 85%). LCMS calculatedfor C₁₆H₃₁N₂O₄ (M+H)⁺: m/z=315.2; found: 315.2.

Intermediate 7. tert-butyl(2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate

Step 1. tert-butyl(2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

To a stirred solution of Intermediate 6 (5.2 g, 16.54 mmol) in CH₂Cl₂(80.0 mL) was added N-(benzyloxy-carbonyloxy)succinimide (4.95 g, 19.85mmol) followed by DIPEA (4.33 mL, 24.81 mmol). The mixture stirred atroom temperature for 2 hours, then diluted with water. The mixture wasextracted with water and brine. The combined organic layers were driedover MgSO₄, filtered, concentrated, and used directly in the next stepwithout further purification.

Step 2. 2-((2S,4S)-4-(((benzyloxy)carbonyl)amino)piperidin-2-yl)aceticacid

The concentrated residue from Step 1 was taken up in CH₂Cl₂ (80.0 mL)and TFA (50.0 mL). The mixture was stirred at room temperature overnightthen concentrated to dryness and used directly in the next step withoutfurther purification.

Step 3.2-((2S,4S)-4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxycarbonyl)piperidin-2-yl)aceticacid

The concentrated residue from Step 2 was taken up in CH₂Cl₂ (80.0 mL)and triethylamine (23.1 mL, 165 mmol) was added slowly. The mixture wasstirred at room temperature for 5 minutes, then Boc-anhydride (4.33 g,19.85 mmol) was added. The mixture was stirred at room temperature foranother 30 minutes. Additional Boc-anhydride was added as necessary.Upon full conversion, the mixture was acidified to pH 4-5 then extractedwith EtOAc. The combined organic layers were dried over MgSO₄, filtered,concentrated, and used directly in the next step without furtherpurification.

Step 4. tert-butyl(2S,4S)-2-(2-amino-2-oxoethyl)-4-(((benzyloxy)carbonyl)amino)piperidine-1-carboxylate

The concentrated residue from Step 3 was taken up in THF (80.0 mL) andDIPEA (8.67 mL, 49.6 mmol). The mixture was cooled to 0° C., thenisobutyl chloroformate (5.43 mL, 41.3 mmol) was added slowly. Themixture was stirred at 0° C. for another 20 minutes before ammoniumhydroxide (28% in water, 23.0 mL, 165 mmol) was added to the mixture.After stirring for another 5 minutes, the mixture was diluted with brineand extracted with EtOAc. The combined organic layers were dried overMgSO₄, filtered, concentrated, and purified by column chromatography0-8% MeOH in DCM.

Step 5. tert-butyl(2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-(cyanomethyl)piperidine-1-carboxylate

The purified product from Step 4 was taken up in THF (80.0 mL) andtriethylamine (6.0 mL, 43 mmol). The mixture was cooled to 0° C., thenTFAA (3.5 mL, 24.8 mmol) was added slowly. The mixture was stirred at 0°C. for another 30 minutes before diluting with EtOAc and extracting withbrine. The combined organic layers were dried over MgSO₄, filtered,concentrated to dryness, and purified on silica gel to yield the desiredproduct (5.12 g, 83% over 5 steps). LCMS calculated for C₁₆H₂₀N₃O₄(M+H-tert-butyl)⁺: m/z=318.1; found: 318.1.

Step 6. tert-butyl(2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate

A round-bottom flask containing a stir bar was charged with tert-butyl(2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-(cyanomethyl)piperidine-1-carboxylate(5.12 g, 13.71 mmol), palladium on carbon (10 wt %, 2.92 g, 2.74 mmol),and MeOH (45 mL). The round-bottom was evacuated and backfilled with H₂(this process was repeated a total of three times) and the mixture wasstirred vigorously at room temperature for 1.5 hours with a balloon ofH₂ attached. The mixture was then filtered over celite and the solidswere washed with EtOAc. The filtrate was concentrated and used directlyin the next step without further purification.

Intermediate 8. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

Step 1. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

DIPEA (5.62 ml, 32 mmol) was added to a solution of7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1)(10 g, 21.5 mmol) and tert-butyl(2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate (Intermediate 7)(5.15 g, 21.5 mmol) in MeCN (100 mL) at room temperature. The reactionwas stirred at 50° C. for 3 h. Upon full conversion, the reactionmixture was concentrated.

The residue was dissolved in MeOH (80 mL), Sodium thiomethoxide (3.0 g,43 mmol) was added at room temperature in one portion, upon fullconversion, the reaction mixture was diluted with water, filtered, andwashed with water to afford brown solid.

The solid was redissolved in MeOH (80 mL), CH₂Cl₂ (20 mL) and aqueousammonium hydroxide (28 mL, 440 mmol). Sodium dithionite (11.2 g, 132mmol) was added in one portion then the reaction was stirred vigorouslyat room temperature. Upon completion, the reaction was quenched with theaddition of H₂O (100 mL), and extracted with CH₂Cl₂ (100 mL). Theorganic phase was washed with H₂O twice, dried over Na₂SO₄, thenconcentrated to afford the desired diamine product as a red viscous oil.

The crude diamine was dissolved in glacial acetic acid (50 mL, 875mmol). Sodium nitrite (2.97 g, 43.0 mmol) was added in one portion. Thereaction was stirred at room temperature for 2 h. Upon completion, thereaction contents were poured into vigorously stirred ice water. Theprecipitated solids were collected via filtration, washed with NaHCO₃,water, and diethyl ether. The solids were then dried under vacuum toprovide the desired product as a brown solid (7.1 g, 50% yield). LCMScalculated for C₂₂H₂₄BrFIN₆O₂S (M+H)⁺: m/z=661.0; found: 660.9.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

A screw-cap vial equipped with a magnetic stir bar was chargedtert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(1.19 g, 1.832 mmol), methylboronic acid (1.096 g, 18.32 mmol),tripotassium phosphate (1.166 g, 5.49 mmol), andbis(triphenylphosphine)-palladium(II) chloride (257 mg, 0.366 mmol)followed by dioxane (10.0 mL) and water (2.0 mL). The vial was sealedwith a Teflon-lined septum, evacuated and backfilled with nitrogen (thisprocess was repeated a total of three times). Then the reaction wasstirred at 90° C. for 3 hours. After cooling to room temperature, themixture was diluted with brine and extracted with EtOAc. The combinedorganic layers were dried over MgSO₄, filtered, concentrated to dryness,and purified on silica gel to yield the desired product. LCMS calculatedfor C₂₃H₂₇BrFN₆O₂S (M+H)⁺: m/z=549.1; found: 549.1.

Example 1a and Example 1b.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A reaction vial was charged with Intermediate 3 (1.3 g, 1.9 mmol), XPhosPd G2 (76 mg, 0.097 mmol), (5-fluoroquinolin-8-yl)boronic acid (408 mg,2.1 mmol), K₃PO₄ (1.24 g, 5.83 mmol) and dioxane (5 mL) and H₂O (1 mL).The mixture was sparged with N₂ for 5 min before heated and stirred at90° C. for 1 h. Upon completion, the reaction was cooled to roomtemperature, diluted with EtOAc (20 mL) and washed with aq. NH₄Cl (20mL). The organic phase was separated, dried over MgSO₄, filtered, thenconcentrated. The crude product was first purified by silica (20 g,50-100% EtOAc in CH₂Cl₂), then further purified using prep-LCMS (XBridgeC18 column, eluting with a gradient of acetonitrile/water containing0.15% NH₄OH, at flow rate of 60 mL/min) to afford the desired product asa pair of diastereomers (white amorphous powder, 21% combined yield).

Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₉F₅N₉O₂ (M+H)⁺m/z=736.3; found 736.2.

Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₉F₅N₉O₂ (M+H)⁺m/z=736.3; found 736.2.

Step 2.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

A reaction vial charged with tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(diastereomer 1, 150 mg, 0.24 mmol) from step 1 was added TFA (1 mL) atroom temperature. After stirring for 15 min, the volatiles were removed.The residue was redissolved in acetonitrile (2 mL) and cooled to 0° C.DIPEA (0.36 mL) was added to the reaction, followed by(E)-4-fluorobut-2-enoic acid (42 mg, 0.41 mmol) and propylphosphonicanhydride solution (50% in EtOAc, 0.25 mL, 0.41 mmol). After stirring at0° C. for 10 min, the reaction was quenched with aq. NaHCO₃ (5 mL)extracted with EtOAc (5 mL), washed with brine, and concentrated. Thecrude product was redissolved in acetonitrile and purified usingprep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford Example 1a (Diastereomer 1) as a TFA salt in the form of a whiteamorphous powder (50 mg free base equivalent, 34% yield). LCMScalculated for C₃₆H₃₄F₆N₉O (M+H)⁺ m/z=722.3; found 722.2. ¹H NMR (500MHz, DMSO-d6) δ 10.80 (s, 1H), 8.87 (dd, J=4.2, 1.7 Hz, 1H), 8.62 (dd,J=8.5, 1.7 Hz, 1H), 8.41 (s, 1H), 7.82 (dd, J=8.0, 5.8 Hz, 1H), 7.70(dd, J=8.5, 4.2 Hz, 1H), 7.65 (dd, J=9.8, 8.0 Hz, 1H), 6.90-6.77 (m,2H), 5.93 (s, 1H), 5.32 (s, 1H), 5.24-5.20 (m, 1H), 5.12 (dd, J=3.6, 1.2Hz, 1H), 4.97 (s, 1H), 4.76 (s, 1H), 4.57 (s, 1H), 4.37-4.21 (m, 2H),3.61 (d, J=12.7 Hz, 1H), 3.39-3.21 (m, 2H), 2.84 (s, 6H), 2.46-2.22 (m,4H), 1.70 (s, 3H).

Example 1b (Diastereomer 2) was prepared using the above procedure,replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(diastereomer 1) from step 1, with tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(diastereomer 2) from step 1. LCMS calculated for C₃₆H₃₄F₆N₉O (M+H)⁺m/z=722.3; found 722.2.

Example 2a and Example 2b.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A reaction vial was charged with Intermediate 3 (100 mg, 0.15 mmol),(2-methoxy-3-methylphenyl)boronic acid (30 mg, 0.18 mmol), Pd(PPh₃)₄ (17mg, 0.015 mmol), K₃PO₄ (95 mg, 0.45 mmol) and dioxane (2.0 mL) and H₂O(0.4 mL). The mixture was sparged with N₂ for 5 min before heated at 90°C. for 1 h. Upon completion, the reaction was cooled to roomtemperature, diluted with EtOAc (5.0 ml) and washed with aq. NH₄Cl (5.0ml). The organic phase was separated, dried over MgSO₄, andconcentrated. The crude product was purified by silica (10 g, 50-100%EtOAc in DCM) to afford the desired product as a pale yellow viscous oil(92 mg, 87% yield). LCMS calculated for C₃₆H₄₃F₄N₈O₃ (M+H)⁺ m/z=711.3;found 711.2.

Step 2.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.Example 2a. Diastereomer 1. Peak 1. LCMS calculated for C₃₅H₃₈F₅NaO₂(M+H)⁺: m/z=697.3; found: 697.3.

Example 2b. Diastereomer 2. Peak 2. LCMS calculated for C₃₅H₃₈F₅N₈O₂(M+H)⁺: m/z=697.3; found: 697.3.

Example 3a and Example 3b.2-((2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 2a and Example 2b, step 1, replacing(2-methoxy-3-methylphenyl)boronic acid with(3-chloro-2-methoxyphenyl)boronic acid. LCMS calculated forC₃₅H₄₀ClF₄N₈O₂ (M+H)⁺: m/z=731.3; found: 731.2.

Step 2.2-((2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.

Example 3a. Diastereomer 1. Peak 1. LCMS calculated for C₃₄H₃₅ClF₅N₈O₂(M+H)⁺: m/z=717.3; found: 717.2.

Example 3b. Diastereomer 2. Peak 2. LCMS calculated for C₃₄H₃₅ClF₅N₈O₂(M+H)⁺: m/z=717.3; found: 717.2.

Example 4a and Example 4b.2-((2S,4S)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate

A sealed tube was charged with Intermediate 4 (3.50 g, 4.75 mmol) andmethyl 2,2-difluoro-2-(fluorosulfonyl)-acetate (1.21 ml, 9.51 mmol)(MFDA), copper(I) iodide (0.272 g, 1.426 mmol) and NMP (20 mL). The tubewas flushed with N₂ then sealed and heated at 80° C. overnight. Thereaction was then cooled to room temperature, poured into NaHCO₃, andextracted with EtOAc. The organic layers were combined, dried overMgSO₄, and concentrated. The crude product was purified on silica (40 g,0-50% EtOAc in Hexanes) to give the desired product (2.5 g, 78% yield).LCMS calculated for C₂₇H₃₃BrF₄N₅O₄S (M+H)⁺: m/z=678.1; found: 678.3.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 3, step 2, replacing tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate.LCMS calculated for C₂₃H₂₄BrF₄N₆O₂S (M+H)⁺: m/z=603.1; found: 603.2.

Step 3. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 1, replacing Intermediate 3 withtert-butyl(2S,4S)-4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₃₂H₂₉F₅N₇O₂S (M+H)⁺: m/z=670.2; found: 670.2.

Step 4. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(85 mg, 0.13 mmol) was dissolved in CH₂Cl₂ (1 mL) and cooled to 0° C.mCPBA (33 mg, 0.19 mmol) was added in one portion, and the reaction wasstirred for 30 min before it was quenched by the addition of saturatedNaHCO₃ (2 mL). The mixture was extracted by CH₂Cl₂ (5 mL) The combinedorganic layers were dried over MgSO₄, filtered, then concentrated toafford a mixture of the crude sulfoxide and sulfone. A vial charged with(S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (16.4 mg, 0.13 mmol) anddry THF (1 mL) was cooled to 0° C. LiHMDS (0.13 mL, 1 M in THF) wasadded. After stirring for 10 min, the reaction was added dropwise to aTHF solution of the crude sulfoxide. After another 10 min of stirring,the reaction was quenched by adding saturated NH₄Cl (5 mL) and extractedwith EtOAc (15 mL). The combined organic layers were dried over MgSO₄and purified on silica (0-100% EtOAc in Hexanes) to give the desiredproduct as a white solid (75 mg, 79% yield). LCMS calculated forC₃₈H₄₀F₅N₈O₃ (M+H)⁺: m/z=751.3; found: 751.5.

Step 5.2-((2S,4S)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.

Example 4a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₅F₆N₈O₂(M+H)⁺: m/z=737.3; found: 737.2.

Example 4b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₅F₆N₈O₂(M+H)⁺: m/z=737.3; found: 737.2.

Example 5a and Example 5b.1-(4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-1-yl)prop-2-en-1-one

Step 1. tert-butyl4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 4a and Example 4b, step 1, replacing Intermediate 4 withIntermediate 5. LCMS calculated for C₂₁H₂₃BrF₄N₅O₂S (M+H)⁺: m/z=564.1;found: 564.0.

Step 2. tert-butyl4-(7-bromo-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 4a and Example 4b, step 4, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl4-(7-bromo-6-fluoro-4-(methylthio)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₂₇H₃₄BrF₄N₆O₃ (M+H)⁺: m/z=645.2; found: 645.4.

Step 3. tert-butyl4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 1, replacing Intermediate 3 withtert-butyl4-(7-bromo-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₆H₃₉F₅N₇O₃ (M+H)⁺: m/z=712.3; found: 712.5.

Step 4.1-(4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-1-yl)prop-2-en-1-one

tert-butyl4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(25 mg, 0.039 mmol) was added TFA (0.5 mL) and stirred at r.t. for 10min. The solvent was removed under vacuum. The residue was dissolved inCH₂Cl₂ (1.0 mL) and cooled to 0° C., to this was added triethylamine (16μL, 0.116 mmol), followed by acryloyl chloride (5.3 mg, 0.058 mmol) andthe reaction was stirred at 0° C. for 20 min. The reaction was dilutedwith CH₂Cl₂, washed with saturated NaHCO₃, and the organic solvent wasdried and concentrated. The crude product was redissolved inacetonitrile and purified using prep-LCMS (XBridge C18 column, elutingwith a gradient of acetonitrile/water containing 0.1% TFA, at flow rateof 60 mL/min).

Example 5a. Diastereomer 1. Peak 1. LCMS calculated for C₃₄H₃₃F₅N₇O₂(M+H)⁺: m/z=666.3; found: 666.4.

Example 5b. Diastereomer 2. Peak 2. LCMS calculated for C₃₄H₃₃F₅N₇O₂(M+H)⁺: m/z=666.3; found: 666.4.

Example 6.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A mixture of Intermediate 8 (400 mg, 0.728 mmol),(2,3-dimethylphenyl)boronic acid (164 mg, 1.092 mmol), potassiumphosphate (464 mg, 2.184 mmol) and Pd(Ph₃P)₄ (84 mg, 0.073 mmol) indioxane (6 mL)/water (1 mL) was evacuated and backfilled with nitrogen(this process was repeated a total of three times). The reaction wasstirred at 105° C. for 1 h. The mixture was diluted with ethyl acetateand washed with water, brine. The organic was filtered, dried andconcentrated. The product was purified by column eluting withHexane/EtOAc (max EtOAc 80%). LCMS calculated for C₃₁H₃₆FN₆O₂S (M+H)⁺m/z=575.3; found 575.3.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

m-CPBA (167 mg, 0.966 mmol) was added to a solution of tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(370.0 mg, 0.644 mmol) in CH₂Cl₂ (5.0 mL) at 0° C. and then the reactionwas stirred at this temperature for 10 min. The reaction was quenched byadding saturated Na₂S₂O₃, diluted with ethyl acetate and washed withsaturated NaHCO₃, brine, filtered, dried and concentrated and the crudewas used in the next step directly. LCMS calculated for C₃₁H₃₆FN₆O₃S(M+H)⁺ m/z=591.3; found 591.3.

Step 3. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

Triethylamine (0.625 mL, 4.48 mmol) was added to a solution oftert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(0.680 g, 1.121 mmol) and N,N,3-trimethylazetidin-3-amine (0.192 g,1.681 mmol) and then stirred at 70° C. for 2 h. The product was purifiedby column eluting with CH₂Cl₂/MeOH (max MeOH 15%). The atropisomers wereseparated by SFC (column, Phenomenex Lux 5 um Cellulose-1, 21.2×250 mm,Mobile Phase 25% MeOH in CO₂ at 70 mL/min isoc) to get two peaks, namedPK1 and PK2. LCMS calculated for C₃₆H₄₆FN₈O₂ (M+H)⁺ m/z=641.4; found641.5.

Step 4.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(PK1 from Step 3) and replacing (E)-4-fluorobut-2-enoic acid with(E)-4-methoxybut-2-enoic acid. LCMS calculated for C₃₆H₄₄FN₈O₂ (M+H)⁺m/z=639.4; found 639.5. ¹H NMR (600 MHz, DMSO) b 8.04 (s, 1H), 7.30-7.21(m, 2H), 7.00 (dd, J=7.4, 1.5 Hz, 1H), 6.81-6.70 (m, 2H), 5.30 (s, 1H),4.96 (s, 1H), 4.74 (d, J=13.8 Hz, 1H), 4.28 (d, J=14.3 Hz, 1H), 4.11 (s,2H), 3.64 (s, 1H), 3.39 (dd, J=17.0, 8.5 Hz, 1H), 3.33 (s, 3H),3.31-3.19 (m, 3H), 2.83 (s, 6H), 2.43 (s, 3H), 2.41 (m, 1H) 2.35 (s,3H), 2.20 (s, 3H), 2.13 (d, J=13.2 Hz, 1H), 1.94 (s, 3H), 1.70 (s, 3H).

Example 7.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole.LCMS calculated for C₃₁H₃₄FN₈O₂S (M+H)⁺ m/z=601.3; found 601.3.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₁H₃₄FN₈O₃S (M+H)⁺ m/z=617.2; found 617.4.

Step 3. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

LiHMDS (1.0 M in THF) (40.7 mg, 0.243 mmol) was added to a solution of(S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (31.4 mg, 0.243 mmol) inTHF (1.0 mL) at 0° C. and stirred for 5 min. The formed solution wasadded to a solution of tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(75.0 mg, 0.122 mmol) in THF (1.0 mL) at 0° C. and then the reaction wasstirred at rt for 1 h. The mixture was diluted with ethyl acetate andwashed with saturated NaHCO₃, water, filtered and concentrated and usedin the next step directly. LCMS calculated for C₃₇H₄₅FN₉O₃ (M+H)⁺m/z=682.4; found 682.5.

Step 4.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₆H₄₀F₂N₉O₂ (M+H)⁺ m/z=668.3; found 668.5. ¹H NMR(600 MHz, DMSO) δ 8.22 (s, 1H), 8.17 (s, 1H), 7.93 (d, J=8.3 Hz, 1H),7.72 (s, 1H), 7.13 (dd, J=8.2 Hz, 1H), 6.92-6.79 (m, 2H), 5.87 (d,J=16.1 Hz, 1H), 5.58 (d, J=7.5 Hz, 1H), 5.31 (d, J=6.4 Hz, 1H), 5.21 (d,J=2.4 Hz, 1H), 5.14 (d, J=2.9 Hz, 2H), 5.00 (s, 1H), 4.76 (d, J=13.8 Hz,1H), 4.32 (d, J=14.2 Hz, 1H), 4.11 (s, 3H), 3.96 (p, J=7.7 Hz, 1H),3.65-3.59 (m, 3H), 3.45-3.23 (m, 2H), 3.19 (m, 1H), 3.14 (s, 3H), 2.44(s, 3H), 2.32-2.16 (m, 2H), 2.11-1.89 (m, 2H), 1.59 (s, 3H).

Example 8.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with(6-methylpyridin-3-yl)boronic acid. LCMS calculated for C₂₉H₃₃FN₇O₂S(M+H)⁺ m/z=562.2; found 562.3.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₂₉H₃₃FN₇O₃S (M+H)⁺ m/z=578.2; found 578.4.

Step 3. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 7 Step 3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₅H₄₄FN₈O₃ (M+H)⁺ m/z=643.4; found 643.5.

Step 4.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₄H₃₉F₂NaO₂ (M+H)⁺ m/z=629.3; found 629.5.

Example 9.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 1, replacing (2,3-dimethylphenyl)boronic acid with1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole.LCMS calculated for C₃₁H₃₄FN₈O₂S (M+H)⁺ m/z=601.3; found 601.1.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₁H₃₄FN₈O₃S (M+H)⁺ m/z=617.3; found 617.3.

Step 3. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 7 Step 3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₇H₄₅FN₉O₃ (M+H)⁺ m/z=682.4; found 682.4.

Step 4.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 6, step 4, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₃₇H₄₃FN₉O₃ (M+H)⁺ m/z=680.4; found 680.3.

Example 10.2-((2S,4S)-4-(6-fluoro-7-(4-fluorophenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 9, steps 1-4, replacing1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazolewith (4-fluorophenyl)boronic acid. LCMS calculated for C₃₅H₄₀F₂N₇O₃(M+H)⁺ m/z=644.3; found 644.4. ¹H NMR (500 MHz, DMSO) δ 10.09-9.21 (s,1H), 8.51-7.69 (s, 1H), 7.53-7.44 (dd, J=8.5, 5.7 Hz, 2H), 7.44-7.36 (t,J=8.9 Hz, 2H), 6.83-6.63 (m, 2H), 5.92-5.72 (m, 1H), 5.66-5.47 (m, 1H),5.41-4.20 (m, 2H), 4.18-4.03 (d, J=2.5 Hz, 2H), 4.00-3.85 (m, 1H),3.69-3.53 (m, 2H), 3.39-3.32 (s, 3H), 3.27-3.10 (m, 5H), 2.55-2.36 (d,J=13.8 Hz, 6H), 2.38-2.23 (m, 2H), 2.19-2.04 (m, 2H), 2.00-1.88 (m, 2H),1.64-1.50 (d, J=6.1 Hz, 3H).

Example 11a and Example 11b.8-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

Step 1. tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 8 Step 2, replacing tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylatewith Intermediate 5. LCMS calculated for C₂₁H₂₆BrFN₅O₂S (M+H)⁺m/z=510.1; found 510.1.

Step 2. tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6 Step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.LCMS calculated for C₂₁H₂₆BrFN₅O₃S (M+H-tBu)⁺ m/z=470.0; found 470.0.

Step 3. tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

To a solution of tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(2.51 g, 4.7 mmol) in THF (16 mL) was added(S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (1.23 g, 9.54 mmol) andDBU (1.44 mL, 9.54 mmol). The mixture was stirred at room temperatureovernight, then diluted with EtOAc and extracted with sat'd NH₄Cl. Thecombined organic layers were dried over MgSO₄, filtered, concentrated,and then purified by column chromatography 0-8% MeOH/DCM. LCMScalculated for C₂₇H₃₇BrFN₆O₃ (M+H)⁺ m/z=591.2; found 591.2.

Step 4.8-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1-(piperidin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

A reaction vial was charged with tert-butyl4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(1.56 g, 2.64 mmol),8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (1.472g, 5.27 mmol), SPhos Pd G4 (315 mg, 0.396 mmol), K₃PO₄ (1.679 g, 7.91mmol) and dioxane (11 mL) and H₂O (2.34 mL). The mixture was spargedwith N₂ for 5 min before heated at 100° C. for 3 h. Upon completion, thereaction was cooled to room temperature, diluted with EtOAc (20 mL) andwashed with aq. NH₄Cl (20 mL). The organic phase was separated, driedover MgSO₄, filtered, then concentrated. The concentrated residue wasredissolved in CH₂Cl₂ (10 mL) and TFA (5 mL) and stirred at roomtemperature for 30 min. The solvent was then removed and the crudeproduct was purified using prep-LCMS (XBridge C18 column, eluting with agradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60mL/min) to afford the desired product as a TFA salt. LCMS calculated forC₃₃H₃₅FN₇O (M+H)⁺ m/z=564.3; found 564.2.

Step 5.8-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

To a solution of8-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1-(piperidin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrilein CH₂Cl₂ (0.4 M) was added triethylamine (5 eq), followed by a solutionof acryloyl chloride (1.5 eq) in CH₂Cl₂ (0.5 M). The mixture was stirredat room temperature for 30 minutes, then quenched by the addition ofMeOH. The mixture was concentrated under reduced pressure then dilutedwith AcN and purified by prepLCMS.

Example 11a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₃₇FN₇O₂(M+H)⁺ m/z=618.3; found 618.2.

Example 11b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₃₇FN₇O₂(M+H)⁺ m/z=618.3; found 618.2. ¹H NMR (600 MHz, DMSO) δ 9.98-9.50 (d,J=10.7 Hz, 1H), 8.56-8.46 (dd, J=8.5, 1.3 Hz, 1H), 8.38-8.24 (m, 2H),8.16-8.10 (dd, J=7.2, 1.4 Hz, 1H), 7.91-7.83 (dd, J=8.3, 7.0 Hz, 1H),7.81-7.72 (dd, J=8.3, 7.2 Hz, 1H), 7.71-7.64 (d, J=7.0 Hz, 1H),6.98-6.84 (dd, J=16.7, 10.5 Hz, 1H), 6.25-6.16 (dd, J=16.8, 2.4 Hz, 1H),5.85-5.77 (m, 1H), 5.77-5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.57-5.44 (m,1H), 4.72-4.24 (m, 1H), 4.01-3.88 (m, 1H), 3.71-3.48 (m, 2H), 3.31-3.19(m, 2H), 3.19-3.11 (d, J=4.8 Hz, 3H), 2.48-2.34 (m, 2H), 2.34-2.03 (m,9H), 2.00-1.86 (m, 2H), 1.64-1.52 (d, J=6.1 Hz, 3H).

Example 12.2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-4-((7-bromo-2-chloro-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate

DIPEA (4.5 mL, 25.4 mmol) was added to a solution of7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline (Intermediate 1)(7.9 g, 16.96 mmol) and tert-butyl(2S,4S)-4-amino-2-(cyanomethyl)piperidine-1-carboxylate (Intermediate 7)(4.87 g, 20.35 mmol) in dioxane (50 mL) at room temperature. The mixturewas stirred at room temperature overnight, then the majority of dioxanewas removed under reduced pressure. To the remaining residue was addedice and water and the slurry was stirred vigorously. The solidprecipitate was collected via filtration and dried under air to yieldthe desired product as a yellow solid (quantitative yield). LCMScalculated for C₂₁H₂₂BrClFIN₅O₄ (M+H)⁺: m/z=668.0; found: 667.8.

Step 2. tert-butyl(2S,4S)-4-((7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate

To a solution of tert-butyl(2S,4S)-4-((7-bromo-2-chloro-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate(2.5 g, 3.74 mmol) in dioxane (20 mL) was added DIPEA (2.61 mL, 14.95mmol) and N,N,3-trimethylazetidin-3-amine dihydrochloride (1.05 g, 5.61mmol). The mixture was stirred at 50° C. overnight, then the majority ofdioxane was removed under reduced pressure. To the remaining residue wasadded ice and water and the slurry was stirred vigorously until a finepowder was formed.

The solid precipitate was collected via filtration and dried under airto yield the desired product (2.7 g, 97% yield). LCMS calculated forC₂₇H₃₅BrFIN₇O₄ (M+H)⁺: m/z=746.1; found: 746.1.

Step 3. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

A mixture of tert-butyl(2S,4S)-4-((7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodo-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate(1.0 g, 1.34 mmol) and iron (450 mg, 8.04 mmol) was dissolved in AcOH(5.0 mL) and heated at 60° C. until the nitro group was fully reduced.The mixture was cooled to r.t. then diluted with EtOAc and filtered overa pad of celite. The filtrate concentrated to yield tert-butyl(2S,4S)-4-((3-amino-7-bromo-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-8-fluoro-6-iodoquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₂₇H₃₇BrFIN₇O₂ (M+H)⁺: m/z=716.1; found: 716.1.

To the concentrated residue was added triethyl orthoformate (0.45 mL,2.68 mmol) and toluene (10 mL). The mixture was heated at 100° C.overnight then cooled to r.t. and concentrated. The residue was purifiedby column chromatography (0-10% MeOH/CH₂Cl₂) to yield the desiredproduct (534 mg, 55% yield). LCMS calculated for C₂₈H₃₅BrFIN₇O₂ (M+H)⁺:m/z=726.1; found: 726.1.

Step 4. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 8 Step 2, replacing tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₂₉H₃₈BrFN₇O₂ (M+H)⁺ m/z=614.2; found 614.3.

Step 5. tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

A mixture of tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(785 mg, 1.277 mmol), (2-chloro-3-methylphenyl)boronic acid (283 mg,1.661 mmol), sodium carbonate (542 mg, 5.11 mmol) and Pd(Ph₃P)₄ (221 mg,0.192 mmol) in dioxane (5.5 mL)/water (1.1 mL) was evacuated andbackfilled with nitrogen (this process was repeated a total of threetimes). The reaction was stirred at 90° C. for 3 h. The mixture wasdiluted with ethyl acetate and washed with water, brine. The organic wasfiltered, dried over MgSO₄, concentrated, and purified by prep LCMS.LCMS calculated for C₃₆H₄₄ClFN₇O₂ (M+H)⁺ m/z=660.3; found 660.3.

Step 6.2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.The product was isolated as a mixture of diastereomers. LCMS calculatedfor C₃₅H₃₉ClF₂N₇O (M+H)⁺ m/z=646.3; found 646.3. ¹H NMR (600 MHz, DMSO)δ 10.66-10.31 (s, 1H), 8.54-8.50 (m, 1H), 7.96-7.82 (d, J=4.8 Hz, 1H),7.51-7.46 (m, 1H), 7.44-7.37 (td, J=7.6, 2.4 Hz, 1H), 7.29-7.17 (d,J=7.6 Hz, 1H), 6.89-6.80 (m, 2H), 5.60-5.39 (m, 1H), 5.36-4.85 (m, 1H),5.24-5.11 (dd, J=46.5, 2.5 Hz, 2H), 4.79-4.19 (m, 5H), 3.72-3.56 (m,1H), 3.49-3.14 (m, 2H), 2.85-2.75 (s, 6H), 2.49-2.03 (m, 10H), 1.71-1.61(s, 3H).

Example 13.2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 12, step 1 followed by step 3. LCMS calculated forC₂₂H₂₂BrClFIN₅O₂ (M+H)⁺ m/z=648.0; found 648.0.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 4, Step 2, replacing tert-butyl(2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₂₃H₂₅BrFIN₅O₂S (M+H)⁺: m/z=660.0; found: 660.0.

Step 3. tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 8 Step 2, replacing tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₂₄H₂₈BrFN₅O₂S (M+H)⁺ m/z=548.1; found 548.1.

Step 4. tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 12, Step 5, replacing tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₃₁H₃₄ClFN₅O₂S (M+H)⁺ m/z=594.2; found 594.3.

Step 5. tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 7, Steps 2-3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate.LCMS calculated for C₃₇H₄₅ClFN₆O₃ (M+H)⁺ m/z=675.3; found 675.3.

Step 6.2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylateand replacing (E)-4-fluorobut-2-enoic acid with 2-fluoroacrylic acid.The product was isolated as a mixture of diastereomers. LCMS calculatedfor C₃₅H₃₈ClF₂N₆O₂ (M+H)⁺ m/z=647.3; found 647.3.

Example 14a and 14b.8-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

Step 1. tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 4, replacing tert-butyl(2S,4S)-4-amino-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylatewith tert-butyl (2R,4S)-4-amino-2-methylpiperidine-1-carboxylate. LCMScalculated for C₂₁H₂₅BrFIN₅O₂S (M+H)⁺: m/z=636.0; found: 636.0.

Step 2. tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

A mixture of tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate(5 g, 7.86 mmol), methylboronic acid (0.941 g, 15.72 mmol), potassiumphosphate (5.00 g, 23.57 mmol) anddichlorobis(triphenylphosphine)-palladium(II) (0.827 g, 1.179 mmol) indioxane (60 mL) and Water (20 mL) was heated to and stirred at 90° C.for 3 h. The mixture was then cooled to room temperature, diluted withAcOEt and water, separated. The aqueous layer was extracted with AcOEtand the combined organic layer was washed with brine, dried over Na₂SO₄,filtered and evaporated. The residue was purified by columnchromatography (0-20% EtOAc in CH₂Cl₂) to give the desired product as abrown solid. LCMS calculated for C₂₂H₂₈BrFN₅O₂S (M+H)⁺ m/z=524.1; found524.1.

Step 3. tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 7, Steps 2-3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate.The residue was purified by column chromatography (20-80% AcOEt in DCM)to give the desired product as a brown solid. LCMS calculated forC₂₈H₃₉BrFN₆O₃ (M+H)⁺ m/z=605.2; found 605.2.

Step 4. tert-butyl(2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

A mixture of tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate(200 mg, 0.330 mmol),8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (184mg, 0.661 mmol), tripotassium phosphate (351 mg, 1.651 mmol) and SPhosPd G4 (79 mg, 0.099 mmol) in dioxane (8 mL) and water (2 mL) was heatedto 90° C. After stirring at the same temperate for 3 h, the mixture wascooled to room temperature, diluted with EtOAc and water, separated. Theaqueous layer was extracted with AcOEt and the combined organic layerwas washed with brine, dried over Na₂SO₄, filtered and evaporated. Theresidue was purified by column chromatography (20-80% EtOAc in CH₂Cl₂)to give the desired product as a brown solid. LCMS calculated forC₃₉H₄₅FN₇O₃ (M+H)⁺ m/z=678.4; found 678.4.

Step 5.8-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

tert-butyl(2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate(400 mg, 0.578 mmol) was dissolved in 5 mL of TFA. The mixture wasstirred at room temperature for 10 min, then the solvent was removed.

The residue was dissolved in acetonitrile (10 mL) and Et₃N (484 μl, 3.47mmol) was added. After stirred at 0° C. for several minutes, the cloudymixture became a clear solution. Acryloyl chloride (94 μl, 1.157 mmol)was added. The mixture was stirred at 0° C. for 30 min, then dilutedwith TFA and purified using prep-LCMS (XBridge C18 column, eluting witha gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60mL/min) to afford the desired product.

Example 14a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₉FN₇O₂(M+H)⁺ m/z=632.3; found 632.3.

Example 14b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₉FN₇O₂(M+H)⁺ m/z=632.3; found 632.3.

¹H NMR (500 MHz, DMSO) δ 9.87 (s, 1H), 8.51 (dd, J=8.5, 1.4 Hz, 1H),8.31 (dd, J=8.3, 1.3 Hz, 1H), 8.21 (s, 1H), 8.14 (dd, J=7.2, 1.3 Hz,1H), 7.87 (dd, J=8.3, 7.1 Hz, 1H), 7.77 (dd, J=8.3, 7.2 Hz, 1H), 7.68(d, J=7.0 Hz, 1H), 6.93-6.87 (m, 1H), 6.16 (dd, J=16.7, 2.4 Hz, 1H),5.95 (s, 1H), 5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.51 (dd, J=10.1, 5.9 Hz,1H), 5.08 (s, 1H), 4.68 (s, 1H), 4.27 (d, J=13.9 Hz, 1H), 3.95 (dd,J=7.8, 7.7 Hz, 1H), 3.68 (s, 1H), 3.57 (dq, J=12.2, 6.2 Hz, 1H), 3.17(m, J=4.9 Hz, 3H), 2.46 (d, J=9.7 Hz, 2H), 2.35-2.26 (m, 2H), 2.22 (s,3H), 2.10 (td, J=11.8, 5.2 Hz, 1H), 1.93 (tq, J=14.3, 6.8 Hz, 2H), 1.58(d, J=6.1 Hz, 4H), 1.51 (d, J=7.0 Hz, 2H).

Example 15a and 15b.2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A mixture of Intermediate 8 (1 g, 1.755 mmol),5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole(0.938 g, 2.63 mmol), tripotassium phosphate (1.862 g, 8.77 mmol) andPd(Ph₃P)₄ (0.203 g, 0.175 mmol) in dioxane (12 mL) and water (6 mL) washeated to and stirred at 100° C. for 2 h. The mixture was then cooled toroom temperature, diluted with AcOEt and water, separated. The aqueouslayer was extracted with EtOAc and the combined organic layer was washedwith brine, dried over Na₂SO₄, filtered and evaporated. The residue waspurified by column chromatography (10-40% EtOAc in CH₂Cl₂) to give thedesired product as a brown solid. LCMS calculated for C₃₇H₄₄FN₈O₃S(M+H)⁺ m/z=699.3; found 699.3.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6, Steps 2-3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatein Step 2 and replacing N,N,3-trimethylazetidin-3-amine withN-ethyl-N-methylazetidin-3-amine dihydrochloride in Step 3. The cruderesidue was purified by column chromatography (20-80% EtOAc in CH₂Cl₂)to give the desired product as a brown solid. LCMS calculated forC₄₂H₅₄FN₁₀O₃ (M+H)⁺ m/z=765.4; found 765.3.

Step 3.2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(378 mg, 0.494 mmol) was dissolved in TFA (5 mL). The mixture wasstirred at 60° C. for 10 min, then the solvent was removed. The residuewas combined with 2-fluoroacrylic acid (131 mg, 1.455 mmol), thenacetonitrile (10 mL) was added followed by DIPEA (506 μl, 2.89 mmol) andT3P (853 μl, 50% in EtOAc, 1.447 mmol). After stirred at roomtemperature overnight, the mixture was diluted with TFA, filtered andpurified using prep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford the desired products as a TFA salt.

Example 15a. Diastereomer 1. Peak 1. LCMS calculated for C₃₅H₃₉F₂N₁₀O(M+H)⁺ m/z=653.3; found 653.3. ¹H NMR (600 MHz, DMSO) δ 13.02 (s, br,1H), 10.21 (s, br, 1H), 8.09 (s, 1H), 7.47 (s, 1H), 7.30 (s, 1H), 5.86(td, J=11.4, 5.5 Hz, 1H), 5.45-5.38 (m, 1H), 5.33 (m, 1H), 4.69 (s, 8H),4.40 (d, J=8.8 Hz, 1H), 4.15 (s, 1H), 3.73 (s, 1H), 3.36 (dd, J=17.2,6.6 Hz, 2H), 3.11 (m, 1H), 2.86 (s, 3H), 2.49 (d, J=9.5 Hz, 1H), 2.47(s, 3H), 2.13 (s, 3H), 2.02 (s, 3H), 1.25 (t, J=7.3 Hz, 3H).

Example 15b. Diastereomer 2. Peak 2. LCMS calculated for C₃₅H₃₉F₂N₁₀O(M+H)⁺ m/z=653.3; found 653.3.

Example 16a and Example 16b.8-(6-fluoro-1-(1-((E)-4-fluorobut-2-enoyl)piperidin-4-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

A round bottom flask was charged with8-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1-(piperidin-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrilefrom Example 11, step 1-4 (600 mg, 0.854 mmol), propylphosphonicanhydride solution (50% in EtOAc, 1.6 mL, 2.56 mmol),(E)-4-fluorobut-2-enoic acid (267 mg, 2.56 mmol), and acetonitrile (15mL). The reaction flask was cooled to 0° C. and TEA (1.7 mL, 12.8 mmol)was added to the reaction. After stirring at r.t. for 2 h, the reactionwas quenched with aq. NaHCO₃ (10 mL) extracted with EtOAc (25 mL×3), andconcentrated. The crude mixture was redissolved in acetonitrile andpurified using prep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford the desired product.

Example 16a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₈F₂N₇O₂(M+H)⁺: m/z=650.3; found: 650.3.

Example 16b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₈F₂N₇O₂(M+H)⁺: m/z=650.3; found: 650.3.

Example 17a and Example 17b.8-(1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

Step 1. tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6, Steps 2-3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith Intermediate 8. The crude residue was purified on silica (0-10%MeOH in DCM) to give the desired product as a brown solid (700 mg, 63%yield). LCMS calculated for C₂₈H₃₇BrFN₈O₂ (M+H)⁺: m/z=615.2; found:615.2.

Step 2. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(8-cyanonaphthalen-1-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A reaction vial was charged with tert-butyl(2S,4S)-4-(7-bromo-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(500 mg, 0.79 mmol), SPhos Pd G4 (97 mg, 0.12 mmol),8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (453mg, 1.63 mmol), K₃PO₄ (571 mg, 2.44 mmol), and dioxane (9 mL) and H₂O (3mL). The mixture was sparged with N₂ for 5 min before heated at 90° C.for 2 h. Upon completion, the reaction was cooled to room temperature,diluted with EtOAc (30 mL) and washed with aq. water (20 mL). Theorganic phase was separated, dried over MgSO₄ and purified on silica(0-10% MeOH in DCM) to give the desired product as a brown solid (358mg, 64% yield). LCMS calculated for C₃₉H₄₃FN₉O₂ (M+H)⁺: m/z=688.4;found: 688.3.

Step 3.8-(1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(8-cyanonaphthalen-1-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate,and replacing (E)-4-fluorobut-2-enoic acid with 2-fluoroacrylic acid.

Example 17a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₆F₂N₉O(M+H)⁺: m/z=660.3; found: 660.3. ¹H NMR (600 MHz, DMSO) δ 8.48 (dd,J=8.4, 1.3 Hz, 1H), 8.28 (dd, J=8.4, 1.3 Hz, 1H), 8.12 (dd, J=7.1, 1.3Hz, 1H), 8.06 (s, 1H), 7.85 (dd, J=8.3, 7.1 Hz, 1H), 7.75 (dd, J=8.3,7.1 Hz, 1H), 7.62 (dd, J=7.1, 1.3 Hz, 1H), 5.90-5.75 (m, 1H), 5.45-5.30(m, 2H), 4.00-4.50 (m, 11H), 2.85 (s, 6H), 2.50 (s, 1H), 2.20-2.15 (m,4H), 1.70 (s, 3H).

Example 17b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₆F₂N₉O(M+H)⁺: m/z=660.3; found: 660.3.

Example 18a and 18b.2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. 7-bromo-6,8-dichloro-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

NCS (5.6 g, 41.9 mmol) was added to a solution of2-amino-4-bromo-3-chlorobenzoic acid (10.0 g, 39.9 mmol) in 1,4-dioxane(100 mL) and then the reaction was stirred at 70° C. overnight. Themixture was cooled to room temperature before triphosgene (4.7 g, 15.96mmol) was added and then stirred at 80° C. for 2 h. Upon completion, icewas added and the mixture was stirred for 30 min. The solid precipitatewas collected via filtration and washed with ice water to provide thedesired product.

Step 2. 7-bromo-6,8-dichloro-3-nitroquinoline-2,4-diol

DIPEA (3.49 mL, 19.97 mmol) was added to a solution of ethyl2-nitroacetate (2.66 g, 19.97 mmol) in toluene (40.0 mL) at roomtemperature. After stirring for 10 min,7-bromo-6,8-dichloro-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (3.1 g, 10.0mmol) was added and the resulting mixture was stirred at 95° C. for 3 h.The reaction was cooled with ice water, then the solids were collectedvia filtration and washed with small amount of ethyl acetate to providethe desired product as a yellow solid (1.05 g, 30%). LCMS calculated forC₉H₄BrCl₂N₂O₄ (M+H)⁺: m/z=352.9; found 353.0.

Step 3. 7-bromo-2,4,6,8-tetrachloro-3-nitroquinoline

DIPEA (0.98 mL, 5.6 mmol) was added to a mixture of7-bromo-6,8-dichloro-3-nitroquinoline-2,4-diol (1.0 g, 2.82 mmol) inPOCl₃ (6.0 mL, 64.4 mmol) at room temperature. The reaction was thenstirred at 100° C. for 3 h. The solvent was removed under vacuum. Theconcentrated residue was redissolved in ethyl acetate then washed withNaHCO₃ (aq.). The combined organic layers were concentrated under vacuumto provide the crude product which was used directly in the next stepwithout further purification.

Step 4. tert-butyl(2S,4S)-4-((7-bromo-6,8-dichloro-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate

To a solution of 7-bromo-2,4,6,8-tetrachloro-3-nitroquinoline (1 g, 2.56mmol) in CH₂Cl₂ (10 mL) was added DIPEA (1.8 mL, 10.24 mmol) andIntermediate 7 (730 mg, 3.0 mmol). The mixture was stirred at r.t.overnight, then N,N,3-trimethylazetidin-3-amine (350 mg, 3.0 mmol) wasadded and the mixture was stirred at r.t. until completion. The reactionwas diluted with CH₂Cl₂ and then washed with saturated NH₄Cl solution.The solvent was removed under vacuum and the crude product was useddirectly in the next step without further purification. LCMS calculatedfor C₂₇H₃₅BrCl₂N₇O₄ (M+H)⁺: m/z=670.1; found 670.1.

Step 5. tert-butyl(2S,4S)-4-(7-bromo-6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

A slurry of tert-butyl(2S,4S)-4-((7-bromo-6,8-dichloro-2-(3-(dimethylamino)-3-methylazetidin-1-yl)-3-nitroquinolin-4-yl)amino)-2-(cyanomethyl)piperidine-1-carboxylate(1.7 g, 2.56 mmol) in THF/MeOH/H₂O (1:1:1) (30 mL), iron (1.43 g, 25.6mmol) and NH₄Cl (1.37 g, 25.6 mmol) was heated at 65° C. for 30 min.Upon completion, the reaction was diluted with MeOH and filtered. Thesolvent was removed under vacuum, the residue was basified with NaHCO₃and extracted with CH₂Cl₂. The solvent was removed under vacuum.

The concentrated residue was redissolved in acetic acid (5 mL) andsodium nitrite (176 mg, 5.12 mmol) was added. The mixture was stirred atroom temperature for 1 hour. Upon completion, the solvent was removedunder vacuum and the residue was basified with NaHCO₃ and extracted withCH₂Cl₂. The combined organic layers were dried over MgSO4, filtered,concentrated to dryness, and purified on silica gel to yield the desiredproduct (1.0 g, 60%). LCMS calculated for C₂₇H₃₄BrCl₂NaO₂ (M+H)⁺:m/z=651.1; found 651.0.

Step 6. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

To a solution of tert-butyl(2S,4S)-4-(7-bromo-6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(650 mg, 1.0 mmol) in dioxane/H₂O=4:1 (10 mL), K₃PO₄ (850 mg, 4 mmol),(5-fluoroquinolin-8-yl)boronic acid (286 mg, 1.5 mmol), and Pd(PPh₃)₄(80 mg, 0.07 mmol) were added, the reaction mixture was heated to 90° C.for 1.5 hours. Upon completion, the reaction was diluted with water andextracted with CH₂Cl₂, the combined organic layers were dried overMgSO₄, filtered, concentrated to dryness, and purified on silica gel toyield the desired product as a mixture of diastereomers (550 mg, 76%).LCMS calculated for C₃₆H₃₉Cl₂FN₉O₂ (M+H)⁺: m/z=718.3; found 718.2.

Step 7.2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 6, step 4, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.The product was purified using prep-LCMS (XBridge C18 column, elutingwith a gradient of acetonitrile/water containing 0.15% NH₄OH, at flowrate of 60 mL/min) to afford desired products.

Example 18a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₃₇Cl₂FN₉O₂(M+H)⁺ m/z=716.2; found 716.2.

Example 18b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₃₇Cl₂FN₉O₂(M+H)⁺ m/z=716.2; found 716.2.

Example 19a and 19b.2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.

Example 19a. Diastereomer 1. Peak 1. LCMS calculated for C₃₅H₃₄Cl₂F₂N₉O(M+H)⁺ m/z=704.2; found 704.2.

Example 19b. Diastereomer 2. Peak 2. LCMS calculated for C₃₅H₃₄Cl₂F₂N₉O(M+H)⁺ m/z=704.2; found 704.2.

Example 20.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 1a and Example 1b, step 2 for Example 1a, replacing(E)-4-fluorobut-2-enoic acid with (E)-4-methoxybut-2-enoic acid.

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A reaction vial was charged with Intermediate 3 (1.3 g, 1.9 mmol), XPhosPd G2 (76 mg, 0.097 mmol), (5-fluoroquinolin-8-yl)boronic acid (408 mg,2.1 mmol), K₃PO₄ (1.24 g, 5.83 mmol) and dioxane (5 mL) and H₂O (1 mL).The mixture was sparged with N₂ for 5 min before heated at 90° C. for 1h. Upon completion, the reaction was cooled to room temperature, dilutedwith EtOAc (20 mL) and washed with aq. NH₄Cl (20 mL). The organic phasewas separated, dried over MgSO₄, filtered, then concentrated. The crudeproduct was first purified by silica (20 g, 50-100% EtOAc in CH₂Cl₂),then further purified using prep-LCMS (XBridge C18 column, eluting witha gradient of acetonitrile/water containing 0.15% NH₄OH, at flow rate of60 mL/min) to separate the diastereomers (white amorphous powder, 21%combined yield).

Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₉F₅N₉O₂ (M+H)⁺m/z=736.3; found 736.2.

Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₉F₅N₉O₂ (M+H)⁺m/z=736.3; found 736.2.

Step 2.2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

A reaction vial charged with tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(Diastereomer 1, 300 mg, 0.41 mmol) from step 1 was added TFA (1 mL) atroom temperature. After stirring for 15 min, the volatiles were removed.The residue was redissolved in acetonitrile (4 mL) and cooled to 0° C.DIPEA (0.21 mL) was added to the reaction, followed by(E)-4-methoxybut-2-enoic acid (71 mg, 0.61 mmol) and propylphosphonicanhydride solution (50% in EtOAc, 0.50 mL, 0.82 mmol). After stirring at0° C. for 10 min, the reaction was quenched with aq. NaHCO₃ (5 mL)extracted with EtOAc (5 mL), washed with brine, and concentrated. Thecrude product was redissolved in acetonitrile and purified usingprep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford Example 20 (single diastereomer) as a TFA salt in the form of awhite amorphous powder. LCMS calculated for C₃₇H₃₇F₅N₉O₂ (M+H)⁺m/z=734.3; found 734.4. ¹H NMR (600 MHz, DMSO-d6) δ 10.80 (s, 1H), 8.87(dd, J=4.2, 1.7 Hz, 1H), 8.62 (dd, J=8.5, 1.8 Hz, 1H), 8.41 (s, 1H),7.82 (dd, J=8.0, 5.9 Hz, 1H), 7.70 (dd, J=8.5, 4.1 Hz, 1H), 7.65 (dd,J=9.7, 8.0 Hz, 1H), 6.81-6.72 (m, 2H), 5.93 (s, 1H), 5.31 (s, 1H), 4.96(m, 2H), 4.76-4.32 (m, 2H), 4.11 (d, J=3.1 Hz, 2H), 3.67-3.34 (m, 2H),3.33 (s, 3H), 3.19 (td, J=16.6, 10.1 Hz, 2H), 2.84 (s, 6H), 2.49-2.43(m, 2H), 2.33 (s, 1H), 2.21 (d, J=15.7 Hz, 1H), 1.71 (s, 3H).

Example 21.2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A reaction vial was charged with tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(315 mg, 0.5 mmol), Bis(pinacolato)diboron (165 mg, 0.65 mmol),Pd(dppf)Cl₂ (36 mg, 0.05 mmol), Potassium acetate (147 mg, 1.5 mmol) anddioxane (5 mL). The mixture was sparged with N₂ for 5 min before heatedat 80° C. for 16 h. Upon completion, the reaction was cooled to roomtemperature, diluted with DCM (100 mL) and washed with aq. NH₄Cl (20mL). The organic phase was separated, dried over MgSO₄, filtered, thenconcentrated. The crude product used for next step without furtherpurification.

Step 2.2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

A reaction vial was charged with crude tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatefrom last step 1, 1-chloro-4-fluoroisoquinoline (118 mg, 0.65 mmol),Pd(PPh₃)₄ (58 mg, 0.05 mmol), K₃PO₄ (318 mg, 1.5 mmol), dioxane (4 mL)and water (1 mL). The mixture was heated at 90° C. for 1 h. Uponcompletion, the reaction was cooled to room temperature, diluted withethyl acetate (100 mL) and washed with aq. NH₄Cl (20 mL). The organicphase was separated, dried over MgSO₄, filtered, then concentrated.

The crude product was dissolved in 5 mL DCM/TFA (1:1) solution. Uponcompletion, the reaction was concentrated. The crude product wasredissolved in acetonitrile and purified using prep-LCMS (XBridge C18column, eluting with a gradient of acetonitrile/water containing 0.1%TFA, at flow rate of 60 mL/min) to afford desired product as a mixtureof diastereomers, LCMS calculated for C₃₃H₃₅F₂N₈O (M+H)⁺ m/z=597.3;found 597.3.

Step 3.2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

A reaction vial charged with2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile(30 mg, 0.05 mmol), DIPEA (0.087 mL, 0.5 mmol), (E)-4-methoxybut-2-enoicacid (12 mg, 0.1 mmol), 1 mL MeCN, and propylphosphonic anhydridesolution (50% in EtOAc, 0.06 mL, 0.1 mmol). After stirring at 0° C. for10 min, the reaction was diluted in acetonitrile and purified usingprep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford desire product as a mixture of diastereomers.

Example 21. LCMS calculated for C₃₈H₄₁F₂NaO₃ (M+H)⁺ m/z=695.3; found695.3.

Example 22.2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 21, replacing (E)-4-methoxybut-2-enoic acid with 2-fluoroacrylicacid.

Example 22. LCMS calculated for C₃₆H₃₆F₃NaO₂ (M+H)⁺ m/z=669.3; found669.3.

Example 23a and Example 23b.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1.3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline

A mixture of 4-bromo-3-methylisoquinoline (1.110 g, 5.0 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (2.285 g,9.00 mmol), potassium acetate (1.472 g, 15.00 mmol) and PdCl₂(dppf)(0.366 g, 0.500 mmol) in Dioxane (20.0 mL) was stirred at 105° C. for 5h. The solvent was removed and the product was purified by columneluting with Hexane/EtOAc (max EtOAc 80%). LCMS calculated forC₁₆H₂₁BNO₂ (M+H)⁺ m/z=270.1; found 270.1.

Step 2.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

A mixture of tert-butyl(2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(45.0 mg, 0.071 mmol),3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinoline(28.8 mg, 0.107 mmol), potassium phosphate (45.4 mg, 0.214 mmol) andMethanesulfonato(2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)(2′-methylamino-1,1′-biphenyl-2-yl)palladium(II)dichloromethane adduct (SPhos Pd G4) (5.68 mg, 7.14 μmol) in Dioxane (2ml)/Water (0.4 ml) was vacuumed and then refilled with N2 twice and thenthe reaction was stirred at 95° C. for 4 h. The mixture was diluted withMeCN and then purified by prep-HPLC under PH=10. The solvent was removedand the residue was dissolved in MeCN (1.0 mL) and then TFA (1.0 mL) wasadded and the reaction was stirred at rt for 40 min. The solvent wasremoved and the crude product was used in the next step directly. LCMScalculated for C₃₄H₃₈FN₈O (M+H)⁺ m/z=593.3; found 593.4.

Step 3.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Triethylamine (5.64 μl, 0.040 mmol) was added to a solution of2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile(4.0 mg, 6.75 μmol), 2-fluoroacrylic acid (1.215 mg, 0.013 mmol) and1-propanephosphonic acid cyclic anhydride (T3P 50% in EtOAc) (4.02 μl,0.013 mmol) in ethyl acetate (0.8 ml) at 0° C. and then the reaction wasstirred for 30 min. The solvent was removed and the crude product wasredissolved in acetonitrile and purified using prep-LCMS (XBridge C18column, eluting with a gradient of acetonitrile/water containing 0.1%TFA, at flow rate of 60 mL/min) to afford the product as TFA salt.

Example 23a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₉F₂NaO₂(M+H)⁺ m/z=665.3; found 665.4.

Example 23b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₉F₂NaO₂(M+H)⁺ m/z=665.3; found 665.4. ¹H NMR (600 MHz, DMSO) δ 9.46 (s, 1H),8.32 (s, 1H), 8.27 (m, 1H), 7.72 (m, 2H), 7.22 (m, 1H), 5.94 (m 1H),5.64 (m, 1H), 5.28-5.37 (m, 2H), 3.96 (q, 1H), 3.63 (m, 3H), 3.54 (m,1H), 3.38 (m, 2H), 3.21 (m, 1H), 3.16 (s, 3H), 2.51 (m, 4H), 2.40 (s,3H), 2.34 (m, 1H), 2.19 (s, 3H), 2.13 (m, 1H), 1.97 (m, 2H), 1.59 (d,3H).

Example 24a and Example 24b.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 3 replacing 2-fluoroacrylic acid with(E)-4-methoxybut-2-enoic acid.

Example 24a. Diastereomer 1. Peak 1. LCMS calculated for C₃₉H₄₄FN₈O₃(M+H)⁺ m/z=691.3; found 691.5.

Example 24b. Diastereomer 2. Peak 2. LCMS calculated for C₃₉H₄₄FN₈O₃(M+H)⁺ m/z=691.3; found 691.5.

Example 25a and Example 25b.2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1.2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 2 replacing3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolinewith (7-fluoro-2-methylquinolin-8-yl)boronic acid. LCMS calculated forC₃₄H₃₇F₂N₈O (M+H)⁺ m/z=611.3; found 611.4.

Step 2.2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 3 replacing2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrilewith2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.

Example 25a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₃₈F₃NaO₂(M+H)⁺ m/z=683.3; found 683.4.

Example 25b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₃₈F₃NaO₂(M+H)⁺ m/z=683.3; found 683.4.

Example 26a and Example 26b.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1. (1-methylisoquinolin-4-yl)boronic acid

A mixture of 4-bromo-1-methylisoquinoline (0.222 g, 1.0 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.457 g,1.800 mmol), potassium acetate (0.294 g, 3.00 mmol) and PdCl2(dppf)(0.073 g, 0.100 mmol) in dioxane (5.0 ml) was stirred at 105° C. for 4h. The product was purified by prep-HPLC under PH=2 (TFA). LCMScalculated for C₁₀H₁₁BNO₂ (M+H)⁺ m/z=188.1; found 188.1.

Step 2.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 2 replacing3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolinewith (1-methylisoquinolin-4-yl)boronic acid. LCMS calculated forC₃₄H₃₈FN₈O (M+H)⁺ m/z=593.3; found 593.4.

Step 3.2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Triethylamine (5.64 μl, 0.040 mmol) was added to a solution of2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile(4.0 mg, 6.75 μmol), (E)-4-methoxybut-2-enoic acid (1.567 mg, 0.013mmol) and 1-propanephosphonic acid cyclic anhydride (T3P 50% in EtOAc)(4.02 μl, 0.013 mmol) in ethyl acetate (0.8 ml) at 0° C. and then thereaction was stirred for 30 min. The solvent was removed and the crudeproduct was redissolved in acetonitrile and purified using prep-LCMS(XBridge C18 column, eluting with a gradient of acetonitrile/watercontaining 0.1% TFA, at flow rate of 60 mL/min) to afford the product asTFA salt.

Example 26a. Diastereomer 1. Peak 1. LCMS calculated for C₃₉H₄₄FN₈O₃(M+H)⁺ m/z=691.4; found 691.5.

Example 26b. Diastereomer 2. Peak 2. LCMS calculated for C₃₉H₄₄FN₈O₃(M+H)⁺ m/z=691.4; found 691.5.

Example 27a and Example 27b.2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1.2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 2 replacing3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolinewith (7-fluoroquinolin-8-yl)boronic acid. LCMS calculated forC₃₃H₃₅F₂N₈O (M+H)⁺ m/z=597.3; found 597.4.

Step 2.2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 3 replacing2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrilewith2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.

Example 27a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₃₆F₃NaO₂(M+H)⁺ m/z=669.3; found 669.4.

Example 27b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₃₆F₃NaO₂(M+H)⁺ m/z=669.3; found 669.4.

Example 28a and Example 28b.2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indazol-3-yl)acetonitrile

Step 1. (4-bromo-1-methyl-1H-indazol-3-yl)methanol

A mixture of methyl 4-bromo-1H-indazole-3-carboxylate (1.2 g, 4.70 mmol)and iodomethane (1.07 g, 7.5 mmol) in THF (20 mL) was added sodiumhydride (0.34 g, 8.47 mmol, 60% dispersion in mineral oil) at 0° C. Thereaction mixture was stirred for 5 minutes and warmed up to roomtemperature at which point it was allowed to stir for overnight. Uponcompletion, reaction mixture was quenched with aq. NH₄Cl (5 mL). Theaqueous phase was extracted with EtOAc (10 mL×3), dried with MgSO₄,filtered, and concentrated. The subsequent crude mixture was dissolvedin THF (20 mL) and cooled to −78° C. at which point LiAlH₄ (11.7 mL,11.7 mmol, 1M in THF) was slowly added by syringe. The reaction mixturewas allowed to warm to 0° C. and stir for additional 15 minutes at whichpoint aq. NH₄Cl (10 mL) was added slowly. Aqueous phase was extractedwith EtOAc (10 mL×3), dried with MgSO₄, filtered, and concentrated. Thecrude mixture was used in the next step without further purification.LCMS calculated for C₉H₁₀BrN₂O (M+H)⁺ m/z=241.0; found 241.0.

Step 2. 2-(4-bromo-1-methyl-1H-indazol-3-yl)acetonitrile

To (4-bromo-1-methyl-1H-indazol-3-yl)methanol (0.67 g, 2.77 mmol) inCH₂Cl₂ (10 mL) cooled at −78° C. was slowly added PBr₃ (0.39 mL, 4.16mmol). The reaction mixture was allowed to stir at the same temperaturefor 1 hour at which point the reaction mixture was carefully quenchedwith aq. NaHCO₃ (5 mL). The aqueous phase was extracted with EtOAc (20mL×3), dried, with MgSO₄, filtered, and concentrated. To the crudemixture was added DMSO (8 mL) followed by NaCN (0.41 g, 8.32 mmol) atroom temperature. The reaction mixture was stirred at 60° C. for 3 hoursand cooled back to room temperature upon completion. Water (20 mL) wasadded to the reaction flask and the desired product was crashed out. Thedesired product was then collected by filtration, washed with water, andair-dried for 4 hours. LCMS calculated for C₁₀H₉BrN₃ (M+H)⁺ m/z=250.0;found 250.0.

Step 3.2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-yl)acetonitrile

To 2-(4-bromo-1-methyl-1H-indazol-3-yl)acetonitrile (0.12 g, 0.48 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.18 g,0.72 mmol), potassium acetate (0.15 g, 1.54 mmol) and PdCl₂(dppf) (0.024g, 0.033 mmol) in dioxane (3 mL) was stirred at 95° C. for 3 hours. Thesolvent was removed and the product was purified by silica gel columneluting with Hexane/EtOAc (max. EtOAc 50%). LCMS calculated forC₁₆H₂₁BN₃O₂ (M+H)⁺ m/z=298.2; found 298.2.

Step 4. tert-butyl(2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indazol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

To a solution of tert-butyl(2R,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate(40 mg, 0.066 mmol) from Example 14a and Example 14b, step 3 indioxane/H₂O=4:1 (2 mL), K₃PO₄ (49 mg, 0.23 mmol),2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-yl)acetonitrile(29 mg, 0.099 mmol), and SPhosPdG₄ (7.9 mg, 0.01 mmol) were added, thereaction mixture was heated to 90° C. for 1.5 hours. Upon completion,the reaction was diluted with water and extracted with EtOAc (2 mL×3),dried with MgSO₄, filtered, and concentrated. The crude mixture waspurified on silica gel eluting with CH₂Cl₂/MeOH (max. MeOH 10%) to yieldthe desired product as a mixture of diastereomers (20 mg, 44%). LCMScalculated for C₃₈H₄₇FN₉O₃ (M+H)⁺: m/z=696.4, found 696.4.

Step 5.2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indazol-3-yl)acetonitrile

This compound was prepared according to Example 14a and Example 14b,Step 5, replacing tert-butyl(2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylatewith tert-butyl(2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indazol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate.The mixture was purified using prep-LCMS (XBridge C18 column, elutingwith a gradient of acetonitrile/water containing 0.1% TFA, at flow rateof 60 mL/min) to afford the desired product.

Example 28a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₄₁FN₉O₂(M+H)⁺ m/z=650.3; found 650.3.

Example 28b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₄₁FN₉O₂(M+H)⁺ m/z=650.3; found 650.3.

Example 29.2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A mixture of Intermediate 8 (2 g, 3.64 mmol),6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole(1.967 g, 5.46 mmol), tripotassium phosphate (3.86 g, 18.20 mmol) andPd(Ph₃P)₄ (0.421 g, 0.364 mmol) in dioxane (25 ml)/Water (12 ml) wasvacuumed and refilled with N₂ twice and then the reaction was stirred at102° C. for 2 h. The mixture was then cooled to room temperature,diluted with AcOEt and water, separated. The aqueous layer was extractedwith EtOAc and the combined organic layer was washed with brine, driedover Na₂SO₄, filtered and evaporated. The residue was purified by columnchromatography (10-40% EtOAc in CH₂Cl₂) to give the desired product as abrown solid. LCMS calculated for C₃₆H₄₁F₂N₈O₃S (M+H)⁺ m/z=703.3; found703.3.

Step 2.2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

mCPBA (77% wet) (0.842 g, 3.76 mmol) was added to a solution oftert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(2.4 g, 3.41 mmol) in CH₂Cl₂ (30 ml) at 0° C. and then the reaction wasstirred at this temperature for 20 min. The reaction was quenched byadding saturated aqueous Na₂S₂O₃ and Na₂CO₃, diluted with CH₂Cl₂ andseparated. The aqueous layer was extracted with CH₂Cl₂. The combinedorganic layer is dried over Na₂SO₄ and concentrated to give a crudemixture of tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylateand the corresponding sulfonyl compound, which was and the crude wasused in the next step directly.

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(and the corresponding sulfonyl compound, 2.4 g, 3.34 mmol) andN-ethyl-N,3-dimethylazetidin-3-amine (0.428 g, 3.34 mmol) were combinedand acetonitrile (33.4 ml) was added. To the suspension was added DIPEA(1.166 ml, 6.68 mmol) and then the mixture was heated to 70° C. andstirred at same temperature for 2 h. The solvent was evaporated to givethe crude product as a brown solid.

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewas dissolved in 10 ml of TFA and heated to 60° C. and stirred at sametemperature for 10 min. Then cooled to room temperature and diluted withacetonitrile, filtered and purified using prep-LCMS (XBridge C18 column,eluting with a gradient of acetonitrile/water containing 0.1% TFA, atflow rate of 60 mL/min) to separate the diastereomers (white amorphouspowder, 53% combined yield).

Diastereomer 1. Peak 1. LCMS calculated for C₃₂H₃₇F₂N₁₀ (M+H)⁺m/z=599.3; found 599.3.

Diastereomer 2. Peak 2. LCMS calculated for C₃₂H₃₇F₂N₁₀ (M+H)⁺m/z=599.3; found 599.3.

Step 3.2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrilebis(2,2,2-trifluoroacetate) (Diastereomer 2, 395 mg, 0.477 mmol) wascombined with 2-fluoroacrylic acid (134 mg, 1.488 mmol), thenacetonitrile (10 mL) was added followed by DIPEA (780 μl, 4.46 mmol) andT3P (877 μl, 50% in AcOEt, 1.488 mmol). After stirred at roomtemperature overnight, the mixture was diluted with TFA, filtered andpurified using prep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford Example 29 (single diastereomer) as a TFA salt in the form of awhite amorphous powder.

Example 29. LCMS calculated for C₃₅H₃₈F₃N₁₀O (M+H)⁺ m/z=671.3; found671.3. ¹H NMR (600 MHz, DMSO) δ 13.28 (s, br, 1H), 10.16 (s, br, 1H),8.12 (s, 1H), 7.47-7.44 (m, 2H), 5.86 (td, J=11.4, 5.5 Hz, 1H),5.45-5.38 (m, 2H), 5.16 (m, 1H), 4.77-4.73 (m, 2H), 4.50-4.36 (m, 4H),3.66-3.44 (m, 2H), 3.36 (m, 2H), 3.11 (m, 1H), 2.81 (s, 3H), 2.49-2.45(m, 2H), 2.17 (s, 3H), 2.03 (s, 3H), 1.71 (s, 3H), 1.27 (t, J=7.3 Hz,3H).

Example 30.2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1. tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

A mixture of Intermediate 8 (2 g, 3.64 mmol),5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole(1.945 g, 5.46 mmol), tripotassium phosphate (3.86 g, 18.20 mmol) andPd(Ph₃P)₄ (0.421 g, 0.364 mmol) in dioxane (25 ml)/Water (12 ml) wasvacuumed and refilled with N₂ twice and then the reaction was stirred at102° C. for 2 h. The mixture was then cooled to room temperature,diluted with AcOEt and water, separated. The aqueous layer was extractedwith EtOAc and the combined organic layer was washed with brine, driedover Na₂SO₄, filtered and evaporated. The residue was purified by columnchromatography (10-40% EtOAc in CH₂Cl₂) to give the desired product as abrown solid. LCMS calculated for C₃₇H₄₄FN₈O₃S (M+H)⁺ m/z=699.3; found699.3.

Step 2.2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

mCPBA (0.962 g, 77% wet, 4.29 mmol) was added to a solution oftert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(2 g, 2.86 mmol) in CH₂Cl₂ (30 ml) at 0° C. and then the reaction wasstirred at this temperature for 20 min. The reaction was quenched byadding saturated aqueous Na₂S₂O₃ and Na₂CO₃, diluted with CH₂Cl₂ andseparated. The aqueous layer was extracted with CH₂Cl₂. The combinedorganic layer is dried over Na₂SO₄ and concentrated to give a crudemixture of tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylateand the corresponding sulfonyl compound, which was and the crude wasused in the next step directly.

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate(and the corresponding sulfonyl compound, 1.5 g, 2.098 mmol),N-ethyl-N,3-dimethylazetidin-3-amine (0.323 g, 2.52 mmol) were combinedand acetonitrile (20 ml) was added. To the suspension was added DIPEA(1.832 ml, 10.49 mmol) and then the mixture was heated to 70° C. andstirred at same temperature for 2 h. The solvent was evaporated to givethe crude product as a brown solid.

tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylateobtained above was dissolved in 10 ml of TFA and heated to 60° C. andstirred at same temperature for 10 min. Then cooled to room temperatureand diluted with acetonitrile, filtered and purified using prep-LCMS(XBridge C18 column, eluting with a gradient of acetonitrile/watercontaining 0.1% TFA, at flow rate of 60 mL/min) to separate thediastereomers (white amorphous powder, 51% combined yield).

Diastereomer 1. Peak 1. LCMS calculated for C₃₃H₄₀FN₁₀ (M+H)⁺ m/z=595.3;found 595.3.

Diastereomer 2. Peak 2. LCMS calculated for C₃₃H₄₀FN₁₀ (M+H)⁺ m/z=595.3;found 595.3.

Step 3.2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrilebis(2,2,2-trifluoroacetate) (Diastereomer 2, 400 mg, 0.486 mmol) and2-fluoroacrylic acid (88 mg, 0.972 mmol) was combined, then acetonitrile(10 mL) was added followed by DIPEA (849 μl, 4.86 mmol) and T3P (573 μl,50% in AcOEt, 0.972 mmol). After stirred at room temperature overnight,the mixture was diluted with TFA, filtered and purified using prep-LCMS(XBridge C18 column, eluting with a gradient of acetonitrile/watercontaining 0.1% TFA, at flow rate of 60 mL/min) to afford Example 30(single diastereomer) as a TFA salt in the form of a white amorphouspowder.

Example 30. LCMS calculated for C₃₆H₄₁F₂N₁₀O (M+H)⁺ m/z=667.3; found667.3. ¹H NMR (600 MHz, DMSO) δ 13.02 (s, br, 1H), 10.18 (s, br, 1H),8.10 (s, 1H), 7.47 (s, 1H), 7.31 (d, J=5.1 Hz, 1H), 5.86 (td, J=11.4,5.5 Hz, 1H), 5.41 (dd, J=18.2, 4.4 Hz, 1H), 5.38-4.62 (m, 9H), 3.73-3.56(m, 2H), 3.36 (dd, J=16.9, 6.8 Hz, 1H), 3.07 (td, J=14.0, 5.2 Hz, 1H),2.81 (s, 3H), 2.49 (s, 3H), 2.48-2.45 (m, 2H), 2.14 (s, 3H), 2.02 (s,3H), 1.71 (s, 3H), 1.27 (t, J=7.3 Hz, 3H).

Example 31a and Example 31b.2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile

Step 1.2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)acetonitrile

This compound was prepared according to Example 28a and Example 28b,Step 3, replacing 2-(4-bromo-1-methyl-1H-indazol-3-yl)acetonitrile with2-(4-bromo-1-methyl-1H-indol-3-yl)acetonitrile. The mixture was purifiedusing silica gel column chromatography eluting with Hexane/EtOAc (max.EtOAc 50%). LCMS calculated for C₁₇H₂₂BN₂O₂ (M+H)⁺ m/z=297.2, found297.2.

Step 2. tert-butyl(2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate

This compound was prepared according to Example 28a and Example 28b,Step 4, replacing2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-yl)acetonitrilewith2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)acetonitrile.The crude mixture was purified on silica gel eluting with CH₂Cl₂/MeOH(max. MeOH 10%) to yield the desired product as a mixture ofdiastereomers. LCMS calculated for C₃₉H₄₈FN₈O₃ (M+H)⁺: m/z=695.4; found695.4.

Step 3.2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile

This compound was prepared according to Example 14a and Example 14b,Step 5, replacing tert-butyl(2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylatewith tert-butyl(2R,4S)-4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylate.The mixture was purified using prep-LCMS (XBridge C18 column, elutingwith a gradient of acetonitrile/water containing 0.1% TFA, at flow rateof 60 mL/min) to afford the desired product.

Example 31a. Diastereomer 1. Peak 1. LCMS calculated for C₃₇H₄₂FN₈O₂(M+H)⁺ m/z=649.3; found 649.3.

Example 31b. Diastereomer 2. Peak 2. LCMS calculated for C₃₇H₄₂FN₈O₂(M+H)⁺ m/z=649.3; found 649.3. ¹H NMR (600 MHz, DMSO) δ 8.17 (s, 1H),7.62 (d, J=8.3 Hz, 1H), 7.48 (s, 1H), 7.37 (dd, J=8.3, 7.1 Hz, 1H), 7.00(d, J=7.1 Hz, 1H), 6.96-6.84 (m, 1H), 6.16 (dd, J=16.6, 2.4 Hz, 1H),5.95-5.85 (m, 1H), 5.73 (dd, J=10.5, 2.4 Hz, 1H), 5.57 (dq, J=12.4, 6.2Hz, 1H), 4.73-4.62 (m, 1H), 4.31-4.20 (m, 1H), 3.97-3.91 (m, 1H), 3.88(s, 3H), 3.65-3.54 (m, 2H), 3.20-3.07 (m, 6H), 2.51-2.45 (m, 2H),2.38-2.25 (m, 2H), 2.24 (s, 3H), 2.15-1.90 (m, 4H), 1.57-1.43 (m, 6H).

Example 32a and Example 32b.2-(4-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile

Step 1. tert-butyl4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate

This compound was prepared according to Example 11a and Example 11b,Step 4, replacing8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile with2-(1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)acetonitrilefrom Example 31a and Example 31b, Step 1. The crude mixture was purifiedon silica gel eluting with CH₂Cl₂/MeOH (max. MeOH 10%) to yield thedesired product as a mixture of diastereomers. LCMS calculated forC₃₈H₄₆FN₈O₃ (M+H)⁺ m/z=681.4, found 681.4.

Step 2.2-(4-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile

This compound was prepared according to Example 14a and Example 14b,Step 5, replacing tert-butyl(2R,4S)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-methylpiperidine-1-carboxylatewith tert-butyl4-(7-(3-(cyanomethyl)-1-methyl-1H-indol-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylate.The mixture was purified using prep-LCMS (XBridge C18 column, elutingwith a gradient of acetonitrile/water containing 0.1% TFA, at flow rateof 60 mL/min) to afford the desired product.

Example 32a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₄₀FN₈O₂(M+H)⁺ m/z=635.3; found 635.3.

Example 32b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₄₀FN₈O₂(M+H)⁺ m/z=635.3; found 635.3. ¹H NMR (600 MHz, DMSO) δ 8.26 (s, 1H),7.61 (d, J=8.4 Hz, 1H), 7.47 (s, 1H), 7.45-7.35 (m, 1H), 7.00 (d, J=7.1Hz, 1H), 6.93 (dd, J=16.7, 104 Hz, 1H), 6.18 (dd, J=16.7, 2.5 Hz, 1H),5.79-5.70 (m, 2H), 5.59-5.50 (m, 1H), 4.62-4.53 (m, 1H), 4.31-4.24 (m,1H), 3.99-3.90 (m, 1H), 3.87 (s, 3H), 3.70-3.11 (m, 10H), 2.51-2.18 (m,8H), 1.58 (d, J=6.1 Hz, 3H).

Example 33a and 33b.2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

Step 1. 7-bromo-2,4,6-trichloro-8-fluoro-3-nitroquinoline

This compound was prepared according to the procedure described inIntermediate 1, replacing NIS with NCS in Step 1. LCMS calculated forC₉H₂BrCl₃FN₂O₂ (M+H)⁺ m/z=372.8; found 372.8.

Step 2. tert-butyl(2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inIntermediate 8, Step 1, replacing7-bromo-2,4-dichloro-8-fluoro-6-iodo-3-nitroquinoline with7-bromo-2,4,6-trichloro-8-fluoro-3-nitroquinoline. LCMS calculated forC₂₂H₂₄BrClFN₆O₂S (M+H)⁺ m/z=569.1; found 569.1.

Step 3. tert-butyl(2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

A mixture of tert-butyl(2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(1 g, 1.755 mmol),5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole(0.938 g, 2.63 mmol), tripotassium phosphate (1.862 g, 8.77 mmol) andPd(Ph₃P)₄ (0.203 g, 0.175 mmol) in dioxane (11.70 ml)/Water (5.85 ml)was vacuumed and refilled with N₂ twice and then the reaction wasstirred at 102° C. for 2 h. The mixture was then cooled to roomtemperature, diluted with AcOEt and water, separated. The aqueous layerwas extracted with EtOAc and the combined organic layer was washed withbrine, dried over Na₂SO₄, filtered and evaporated. The residue waspurified by column chromatography (10-40% EtOAc in CH₂Cl₂) to give thedesired product as a brown solid. LCMS calculated for C₃₆H₄₁ClFN₈O₃S(M+H)⁺ m/z=719.3; found 719.3.

Step 4. tert-butyl(2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate

This compound was prepared according to the procedure described inExample 6, Steps 2-3, replacing tert-butyl(2S,4S)-2-(cyanomethyl)-4-(7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidine-1-carboxylatewith tert-butyl(2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-4-(methylthio)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylatein Step 2 and replacing N,N,3-trimethylazetidin-3-amine withN-ethyl-N,3-dimethylazetidin-3-amine in Step 3. The crude residue waspurified by column chromatography (20-80% EtOAc in CH₂Cl₂) to give thedesired product as a brown solid. LCMS calculated for C₄₂H₅₃ClFN₁₀O₃(M+H)⁺ m/z=799.4; found 799.4.

Step 5.2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

tert-butyl(2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate(427 mg, 0.534 mmol) was dissolved in TFA (5 mL). The mixture wasstirred at 60° C. for 10 min, then the solvent was removed.

The residue was combined with 2-fluoroacrylic acid (144 mg, 1.601 mmol),then acetonitrile (10 mL) was added followed by DIPEA (0.93 mL, 5.34mmol) and T3P (0.94 mL, 50% in AcOEt, 1.601 mmol). After stirred at roomtemperature overnight, the mixture was diluted with TFA, filtered andpurified using prep-LCMS (XBridge C18 column, eluting with a gradient ofacetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) toafford the desired products as a TFA salt.

Example 33a. Diastereomer 1. Peak 1. LCMS calculated for C₃₅H₃₈ClF₂N₁₀O(M+H)⁺ m/z=687.3; found 687.3.

Example 33b. Diastereomer 2. Peak 2. LCMS calculated for C₃₅H₃₈ClF₂N₁₀O(M+H)⁺ m/z=687.3; found 687.3.

Example 34a and Example 34b.2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

Step 1.2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 23a and Example 23b, step 2 replacing3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolinewith (2-fluoro-6-methoxyphenyl)boronic acid. LCMS calculated forC₃₁H₃₆F₂N₇O₂ (M+H)⁺ m/z=576.3; found 576.4.

Step 2.2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 26a and Example 26b, step 3 replacing2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrilewith2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-2-yl)acetonitrile.

Example 34a. Diastereomer 1. Peak 1. LCMS calculated for C₃₆H₄₂F₂N₇O₄(M+H)⁺ m/z=674.3; found 674.3.

Example 34b. Diastereomer 2. Peak 2. LCMS calculated for C₃₆H₄₂F₂N₇O₄(M+H)⁺ m/z=674.3; found 674.3.

Example 35a and 35b.2-((2S,4S)-4-(8-chloro-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile

This compound was prepared according to the procedure described inExample 33, replacing5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazolewith6-fluoro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazolein Step 3.

Example 35a. Diastereomer 1. Peak 1. LCMS calculated for C₃₄H₃₅ClF₃N₁₀O(M+H)⁺ m/z=691.3; found 691.3.

Example 35b. Diastereomer 2. Peak 2. LCMS calculated for C₃₄H₃₅ClF₃N₁₀O(M+H)⁺ m/z=691.3; found 691.3.

Example A. GDP-GTP Exchange Assay

The inhibitor potency of the exemplified compounds was determined in afluorescence based guanine nucleotide exchange assay, which measures theexchange of bodipy-GDP (fluorescently labeled GDP) for GppNHp(Non-hydrolyzable GTP analog) to generate the active state of KRAS inthe presence of SOS1 (guanine nucleotide exchange factor). Inhibitorswere serially diluted in DMSO and a volume of 0.1 μL was transferred tothe wells of a black low volume 384-well plate. 5 μL/well volume ofbodipy-loaded KRAS G12C diluted to 5 nM in assay buffer (25 mM Hepes pH7.5, 50 mM NaCl, 10 mM MgCl2 and 0.01% Brij-35) was added to the plateand pre-incubated with inhibitor for 2 hours at ambient temperature.Appropriate controls (enzyme with no inhibitor or with a G12C inhibitor(AMG-510)) were included on the plate. The exchange was initiated by theaddition of a 5 μL/well volume containing 1 mM GppNHp and 300 nM SOS1 inassay buffer. The 10 μL/well reaction concentration of the bodipy-loadedKRAS G12C, GppNHp, and SOS1 were 2.5 nM, 500 uM, and 150 nM,respectively. The reaction plates were incubated at ambient temperaturefor 2 hours, a time estimated for complete GDP-GTP exchange in theabsence of inhibitor. For the KRAS G12D and G12V mutants, similarguanine nucleotide exchange assays were used with 2.5 nM as finalconcentration for the bodipy loaded KRAS proteins and with 4 hours and 3hours incubation after adding GppNHp-SOS1 mixture for G12D and G12Vrespectively. A cyclic peptide described to selectively bind G12D mutant(Sakamoto et al., BBRC 484.3 (2017), 605-611) or internal compounds withconfirmed binding were used as positive controls in the assay plates.Fluorescence intensities were measured on a PheraStar plate readerinstrument (BMG Labtech) with excitation at 485 nm and emission at 520nm.

Either GraphPad prism or Genedata Screener SmartFit was used to analyzethe data. The IC₅₀ values were derived by fitting the data to a fourparameter logistic equation producing a sigmoidal dose-response curvewith a variable Hill coefficient.

The KRAS_G12C exchange assay IC₅₀ data, KRAS_G12C pERK assay IC₅₀ data,KRAS_G12C WB pERK assay IC₅₀ data are provided in Table 1 below. Thesymbol “†” indicates IC₅₀ ≤50 nM, “††” indicates IC₅₀ >50 nM but ≤100nm; and “†††” indicates IC₅₀ is >100 nM but ≤1000 nM, “††††” indicatesIC₅₀ is >1 μM but ≤5 μM; “†††††” indicates IC₅₀ is >5 μM “NA” indicatesthat data is not available.

TABLE 1 Ex. No. G12C_exchange G12C_pERK G12C_WB_pERK  1a † † †  1b † ††NA  2a † † †  2b † †† NA  3a † † †††  3b † † NA  4a † † †  4b † † NA  5a† † ††  5b † †† NA  6 † † †††  7 † † †  8 † † †  9 † † † 10 † † † 11a †††† NA 11b † † † 12 † † † 13 † † ††† 14a †† NA NA 14b † † † 15a † † ††15b ††† NA NA 16a † ††† NA 16b † † †† 17a † † †† 17b ††† NA NA 18a † †††† 18b † ††† NA 19a † † † 19b † † NA 20 † † ††† 21 † † † 22 † † † 23a†† NA NA 23b † † † 24a † †† NA 24b † † † 25a † † †† 25b † † NA 26a † ††† 26b † † †† 27a † † †† 27b † † †† 28a ††† NA NA 28b † † †† 29 † † †††30 † † †† 31a ††† NA NA 31b † † †† 32a †† NA NA 32b † † † 33a † † †† 33b††† NA NA 34a † † NA 34b † † † 35a † † †† 35b ††† NA NA

Example B: Luminescent Viability Assay

MIA PaCa-2 (KRAS G12C; ATCC@ CRL-1420), NCI-H358 (KRAS G12C;ATCC@CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC®CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC®CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are cultured inRPMI 1640 media supplemented with 10% FBS (Gibco/Life Technologies).Eight hundred cells per well in RPMI 1640 media supplemented with 2% FBSare seeded into white, clear bottomed 384-well Costar tissue cultureplates containing 50 nL dots of test compounds (final concentration is a1:500 dilution, with a final concentration in 0.2% DMSO). Plates areincubated for 3 days at 370C, 5% CO2. At the end of the assay, 25ul/well of CellTiter-Glo reagent (Promega) is added. Luminescence isread after 15 minutes with a PHERAstar (BMG). Data are analyzed inGenedata Screener using SmartFit for IC₅₀ values.

Example C: Cellular pERK HTRF Assay

MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC®CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC®CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC@CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are purchased fromATCC and maintained in RPMI 1640 media supplemented with 10% FBS(Gibco/Life Technologies). The cells are plated at 5000 cells per well(8 uL) into Greiner 384-well low volume, flat-bottom, and tissue culturetreated white plates and incubated overnight at 370C, 5% CO2. The nextmorning, test compound stock solutions are diluted in media at 3× thefinal concentration and 4 uL are added to the cells, with a finalconcentration of 0.1% of DMSO. The cells are incubated with the testcompounds for 4 hours (G12C and G12V) or 2 hrs (G12D) at 37° C., 5% CO2.Four uL of 4× lysis buffer with blocking reagent (Cisbio) are added toeach well and plates are rotated gently (300 rpm) for 30 minutes at roomtemperature. Four uL per well of Cisbio anti Phospho-ERK 1/2 d2 is mixedwith anti Phospho-ERK 1/2 Cryptate (1:1), and added to each well,incubated overnight in the dark at room temperature. Plates are read onthe Pherastar plate reader at 665 nm and 620 nm wavelengths. Data areanalyzed in Genedata Screener using SmartFit for IC₅₀ values.

Example D: Whole Blood pERK1/2 HTRF Assay

MIA PaCa-2 cells (KRAS G12C; ATCC@ CRL-1420), HPAF-II (KRAS G12D;ATCC@CRL-1997) and YAPC (KRAS G12V; DSMZ ACC382) are maintained in RPMI1640 with 10% FBS (Gibco/Life Technologies). For MIA PaCa-2 assay, cellsare seeded into 96 well tissue culture plates (Corning #3596) at 25000cells per well in 100 uL media and cultured for 2 days at 37° C., 5% CO₂before the assay. For HPAF-II and YAPC assay, cells are seeded in 96well tissue culture plates at 50000 cells per well in 100 uL media andcultured for 1 day before the assay. Whole Blood are added to the 1 uLdots of compounds (prepared in DMSO) in 96 well plates and mixed gentlyby pipetting up and down so that the concentration of the compound inblood is 1× of desired concentration, in 0.5% DMSO. The media isaspirated from the cells and 50 uL per well of whole blood with testcompound is added and incubated for 4 hours for MIA PaCa and YAPC assay;or 2 hours for HPAF-II assay, respectively at 37° C., 5% CO₂. Afterdumping the blood, the plates are gently washed twice by adding PBS tothe side of the wells and dumping the PBS from the plate onto a papertowel, tapping the plate to drain well. Fifty ul/well of 1× lysis buffer#1 (Cisbio) with blocking reagent (Cisbio) and Benzonase nuclease (SigmaCat #E1014-5KU, 1:10000 final concentration) is then added and incubatedat room temperature for 30 minutes with shaking (250 rpm). Followinglysis, 16 uL of lysate is transferred into 384-well Greiner small volumewhite plate using an Assist Plus (Integra Biosciences, NH). Four uL of1:1 mixture of anti Phospho-ERK 1/2 d2 and anti Phospho-ERK 1/2 Cryptate(Cisbio) is added to the wells using the Assist Plus and incubated atroom temperature overnight in the dark. Plates are read on the Pherastarplate reader at 665 nm and 620 nm wavelengths. Data are analyzed inGenedata Screener using SmartFit for IC₅₀ values.

Example E: Ras Activation Elisa

The 96-Well Ras Activation ELISA Kit (Cell Biolabs Inc; #STA441) usesthe Raf1 RBD (Rho binding domain) bound to a 96-well plate toselectively pull down the active form of Ras from cell lysates. Thecaptured GTP-Ras is then detected by a pan-Ras antibody andHRP-conjugated secondary antibody.

MIA PaCa-2 (KRAS G12C; ATCC@ CRL-1420), NCI-H358 (KRAS G12C;ATCC@CRL-5807), A427 (KRAS G12D; ATCC@ HTB53), HPAFII (KRAS G12D; ATCC®CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC®CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are maintained inRPMI 1640 with 10% FBS (Gibco/Life Technologies). The cells are seededinto 96 well tissue culture plates (Corning #3596) at 25000 cells perwell in 100 uL media and cultured for 2 days at 37° C., 5% CO2 so thatthey are approximately 80% confluent at the start of the assay. Thecells are treated with compounds for either 4 hours or overnight at 37°C., 5% CO₂. At the time of harvesting, the cells are washed with PBS,drained well and then lysed with 50 uL of the 1× Lysis buffer (providedby the kit) plus added Halt Protease and Phosphatase inhibitors (1:100)for 1 hour on ice.

The Raf-1 RBD is diluted 1:500 in Assay Diluent (provided in kit) and100 μL of the diluted Raf-1 RBD is added to each well of the Raf-1 RBDCapture Plate. The plate is covered with a plate sealing film andincubated at room temperature for 1 hour on an orbital shaker. The plateis washed 3 times with 250 μL 1× Wash Buffer per well with thoroughaspiration between each wash. 50 μL of Ras lysate sample (10-100 μg) isadded per well in duplicate. A “no cell lysate” control is added in acouple of wells for background determination. 50 μL of Assay Diluent isadded to all wells immediately to each well and the plate is incubatedat room temperature for 1 hour on an orbital shaker. The plate is washed5 times with 250 μL 1× Wash Buffer per well with thorough aspirationbetween each wash. 100 μL of the diluted Anti-pan-Ras Antibody is addedto each well and the plate is incubated at room temperature for 1 houron an orbital shaker. The plate is washed 5 times as previously. 100 μLof the diluted Secondary Antibody, HRP Conjugate is added to each welland the plate is incubated at room temperature for 1 hour on an orbitalshaker. The plate is washed 5 times as previously and drained well. 100μL of Chemiluminescent Reagent (provided in the kit) is added to eachwell, including the blank wells. The plate is incubated at roomtemperature for 5 minutes on an orbital shaker before the luminescenceof each microwell is read on a plate luminometer. The % inhibition iscalculated relative to the DMSO control wells after a background levelof the “no lysate control” is subtracted from all the values. IC₅₀determination is performed by fitting the curve of inhibitor percentinhibition versus the log of the inhibitor concentration using theGraphPad Prism 7 software.

Example F: Inhibition of RAS-RAF and PI3K-AKT Pathways

The cellular potency of compounds was determined by measuringphosphorylation of KRAS downstream effectorsextracellular-signal-regulated kinase (ERK), ribosomal S6 kinase (RSK),AKT (also known as protein kinase B, PKB) and downstream substrate S6ribosomal protein.

To measure phosphorylated extracellular-signal-regulated kinase (ERK),ribosomal S6 kinase (RSK), AKT and S6 ribosomal protein, cells (detailsregarding the cell lines and types of data produced are further detailedin Table 2) were seeded overnight in Corning 96-well tissue culturetreated plates in RPMI medium with 10% FBS at 4×104 cells/well. Thefollowing day, cells were incubated in the presence or absence of aconcentration range of test compounds for 4 hours at 37° C., 5% CO₂.Cells were washed with PBS and lysed with 1× lysis buffer (Cisbio) withprotease and phosphatase inhibitors (Thermo Fisher, 78446). Ten ortwenty μg of total protein lysates was subjected to SDS-PAGE andimmunoblot analysis using following antibodies:phospho-ERK1/2-Thr202/Tyr204 (#9101L), total-ERK1/2 (#9102L),phosphor-AKT-Ser473 (#4060L), phospho-p90RSK-Ser380 (#11989S) andphospho-S6 ribosomal protein-Ser235/Ser236 (#2211S) are from CellSignaling Technologies (Danvers, Mass.).

TABLE 2 KRAS Cell Line Histology alteration Readout H358 Lung G12C pERK,pAKT, p-S6, p-p90RSK MIA PaCa-2 Pancreas G12C pERK, pAKT, p-S6, p-p90RSKHPAF II Pancreas G12D pERK, pAKT, p-S6, p-p90RSK A427 Lung G12D pERK,pAKT, p-S6, p-p90RSK AGS Stomach G12D pERK, pAKT, p-S6, p-p90RSK PaTu8988s Pancreas G12V pERK, pAKT, p-S6, p-p90RSK H441 Lung G12V pERK,pAKT, p-S6, p-p90RSK YAPC Pancreas G12V pERK, pAKT, p-S6, p-p90RSK SW480Colorectal G12V pERK, pAKT, p-S6, p-p90RSK

Example G: In Vivo Efficacy Studies

Mia-Paca-2 (KRAS G12C), H358 (KRAS G12C), HPAF-II (KRAS G12D), AGS (KRASG12D), SW480 (KRAS G12V) or YAPC(KRAS G12V) human cancer cells areobtained from the American Type Culture Collection and maintained inRPMI media supplemented with 10% FBS. For efficacy studies experiments,5×106 cells are inoculated subcutaneously into the right hind flank of6- to 8-week-old BALB/c nude mice (Charles River Laboratories,Wilmington, Mass., USA). When tumor volumes are approximately 150-250mm3, mice are randomized by tumor volume and compounds are orallyadministered. Tumor volume is calculated using the formula (L×W2)/2,where L and W refer to the length and width dimensions, respectively.Tumor growth inhibition is calculated using the formula (1−(VT/VC))×100,where VT is the tumor volume of the treatment group on the last day oftreatment, and VC is the tumor volume of the control group on the lastday of treatment. Two-way analysis of variance with Dunnett's multiplecomparisons test is used to determine statistical differences betweentreatment groups (GraphPad Prism). Mice are housed at 10-12 animals percage, and are provided enrichment and exposed to 12-hour light/darkcycles. Mice whose tumor volumes exceeded limits (10% of body weight)are humanely euthanized by CO₂ inhalation. Animals are maintained in abarrier facility fully accredited by the Association for Assessment andAccreditation of Laboratory Animal Care, International. All of theprocedures are conducted in accordance with the US Public Service Policyon Human Care and Use of Laboratory Animals and with Incyte Animal Careand Use Committee Guidelines.

Example H: Caco2 Assay

Caco-2 cells were grown at 37° C. in an atmosphere of 5% CO₂ in DMEMgrowth medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v)nonessential amino acids, penicillin (100 U/mL), and streptomycin (100μg/mL). Confluent cell monolayers were subcultured every 7 days or 4days for Caco-2 by treatment with 0.05% trypsin containing 1 μM EDTA.Caco-2 cells were seeded in 96-well Transwell plates. The seedingdensity for Caco-2 cells was 14,000 cells/well. DMEM growth medium wasreplaced every other day after seeding. Cell monolayers were used fortransport assays between 22 and 25 days for Caco-2 cells.

Cell culture medium was removed and replaced with HBSS. To measure theTEER, the HBSS was added into the donor compartment (apical side) andreceiver compartment (basolateral side). The TEER was measured by usinga REMS Autosampler to ensure the integrity of the cell monolayers.Caco-2 cell monolayers with TEER values ≥300 Ω·cm² were used fortransport experiments. To determine the P_(app) in the absorptivedirection (A-B), solution of test compound (50 μM) in HBSS was added tothe donor compartment (apical side), while HBSS solution with 4% BSA wasadded to the receiver compartment (basolateral side). The apical volumewas 0.075 mL, and the basolateral volume was 0.25 mL. The incubationperiod was 120 minutes at 37° C. in an atmosphere of 5% CO₂. At the endof the incubation period, samples from the donor and receiver sides wereremoved and an equal volume of acetonitrile was added for proteinprecipitation. The supernatants were collected after centrifugation(3000 rpm, Allegra X-14R Centrifuge from Beckman Coulter, Indianapolis,Ind.) for LCMS analysis. The permeability value was determined accordingto the equation:

P _(app) (cm/s)=(F*VD)/(SA*MD),

where the flux rate (F, mass/time) is calculated from the slope ofcumulative amounts of compound of interest on the receiver side, SA isthe surface area of the cell membrane, VD is the donor volume, and MD isthe initial amount of the solution in the donor chamber.

The Caco-2 permeability assay data are provided in Table 3 below. Thesymbol “†” indicates Caco2≤0.5; and “††” indicates Caco-2 is >0.5 but≤1; and “†††” indicates Caco-2 is >1. “NA” indicates that Caco-2 data isnot available.

TABLE 3 Ex. No. Caco-2  1a †††  2a †††  3a ††  4a ††  5a †††  6 †††  7†††  8 ††  9 ††† 10 ††† 11b ††† 12 †† 13 ††† 14b ††† 15a † 16b ††† 17a††† 18a ††† 19a ††† 20 ††† 21 ††† 22 ††† 23b ††† 24b ††† 25a ††† 26b †††27a ††† 28b ††† 29 ††† 30 ††† 31b ††† 32b ††† 33a †† 34b ††† 35a †

The Caco-2 permeability assay data for certain compounds from WO2021/142252 is provided in Table 4.

TABLE 4 Ex. No. from WO 2021/142252 Caco-2 203a † 198a † 202a †  80a †150a †† 185a † 189 † 193a † 194a † 197a † 200a † 266a †

Example I: Human Whole Blood Stability

The whole blood stability of the exemplified compounds was determined byLC-MS/MS. The 96-Well Flexi-Tier™ Block (Analytical Sales & Services,Inc, Flanders, N.J.) is used for the incubation plate containing 1.0 mLglass vials with 0.5 mL of blood per vial (pooled gender, human wholeblood sourced from BIOIVT, Hicksville, N.Y. or similar). Blood ispre-warmed in water bath to 37° C. for 30 minutes. 96-deep well analysisplate is prepared with the addition of 100 μL ultrapure water/well. 50μL chilled ultrapure water/well is added to 96-deep well samplecollection plate and covered with a sealing mat. 1 μL of 0.5 mM compoundworking solution (DMSO:water) is added to the blood in incubation plateto reach final concentrations of 1 μM, mixed by pipetting thoroughly and50 μL is transferred 50 into the T=0 wells of the sample collectionplate. Blood is allowed to sit in the water for 2 minutes and then 400μL stop solution/well is added (acetonitrile containing an internalstandard). The incubation plate is placed in the Incu-Shaker CO₂ Miniincubator (Benchmark Scientific, Sayreville, N.J.) at 37° C. withshaking at 150 rpm. At 1, 2 and 4-hr, the blood samples are mixedthoroughly by pipetting and 50 μL is transferred into the correspondingwells of the sample collection plate. Blood is allowed to sit in thewater for 2 minutes and then 400 μL of stop solution/well is added. Thecollection plate is sealed and vortexed at 1700 rpm for 3 minutes(VX-2500 Multi-Tube Vortexer, VWR International, Radnor, Pa.), andsamples are then centrifuged in the collection plate at 3500 rpm for 10minutes (Allegra X-14R Centrifuge Beckman Coulter, Indianapolis, Ind.).100 μL of supernatant/well is transferred from the sample collectionplate into the corresponding wells of the analysis plate. The finalplate is vortexed at 1700 rpm for 1 minute and analyze samples byLC-MS/MS. The peak area ratio of the 1, 2, and 4 hr samples relative toT=0 is used to determine the percent remaining. The natural log of thepercent remaining versus time is used determine a slope to calculate thecompounds half-life in blood (t_(1/2)=0.693/slope).

The human whole blood stability data is provided in Table 5 below. Thesymbol “†” indicates WBS ≤70%; “††” indicates WBS >70% but ≤90%; and“†††” indicates WBS >90%. “NA” indicates that WBS data is not available.

TABLE 5 Ex. No. Human whole blood stability at 4 hr  1a †††  2a ††  3a††  4a †††  5a ††  6 NA  7 †††  8 ††  9 NA 10 ††† 11b †† 12 ††† 13 ††14b ††† 15a ††† 16b ††† 17a NA 18a ††† 19a †† 20 ††† 21 ††† 22 NA 23b NA24b NA 25a NA 26b †† 27a NA 28b †† 29 ††† 30 ††† 31b †† 32b †† 33a ††34b ††† 35a †††

Example J: In Vitro Intrinsic Clearance Protocol

For in vitro metabolic stability experiments, test compounds areincubated with human liver microsomes at 37° C. The incubation mixturecontains test compounds (1 μM), NADPH (2 mM), and human liver microsomes(0.5 mg protein/mL) in 100 mM phosphate buffer (pH 7.4). The mixture ispre-incubated for 2 min at 37° C. before the addition of NADPH.Reactions are commenced upon the addition of NADPH and quenched withice-cold methanol at 0, 10, 20, and 30 min. Terminated incubationmixtures are analyzed using LC-MS/MS system. The analytical systemconsisted of a Shimadzu LC-30AD binary pump system and SIL-30ACautosampler (Shimadzu Scientific Instruments, Columbia, Md.) coupledwith a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems(Foster City, Calif.). Chromatographic separation of test compounds andinternal standard is achieved using a Hypersil Gold C18 column (50×2.1mm, 5 μM, 175 Δ) from ThermoFisher Scientific (Waltham, Mass.). Mobilephase A consists of 0.1% formic acid in water, and mobile phase Bconsists of 0.1% formic acid in acetonitrile. The total LC-MS/MS runtimecan be 2.75 minutes with a flow rate of 0.75 mL/min. Peak areaintegrations and peak area ratio calculations are performed usingAnalyst software (version 1.6.3) from Applied Biosystems.

The in vitro intrinsic clearance, CL_(int,in vitro), is calculated fromthe t_(1/2) of test compound disappearance as CL_(int, in vitro)=(0.6931t_(1/2))×(1/C_(protein)), where C_(protein) is the protein concentrationduring the incubation, and t_(1/2) is determined by the slope (k) of thelog-linear regression analysis of the concentration versus timeprofiles; thus, t_(1/2)=ln 2/k. The CL_(int,in vitro) values are scaledto the in vivo values for human by using physiologically based scalingfactors, hepatic microsomal protein concentrations (45 mg protein/gliver), and liver weights (21 g/kg body weight). The equationCL_(int)=CL_(int,in vitro)×(mg protein/g liver weight)×(g liverweight/kg body weight) is used. The in vivo hepatic clearance (CL_(H))is then calculated by using CL_(int) and hepatic blood flow, Q (20mL·min⁻¹kg⁻¹ in humans) in the well-stirred liver model disregarding allbinding from CL_(H)=(Q×CL_(int))/(Q+CL_(int)). The hepatic extractionratio was calculated as CL_(H) divided by Q.

Example K: In Vivo Pharmacokinetics Protocol

For in vivo pharmacokinetic experiments, test compounds are administeredto male Sprague Dawley rats or male and female Cynomolgus monkeysintravenously or via oral gavage. For intravenous (IV) dosing, testcompounds are dosed at 0.5 to 1 mg/kg using a formulation of 10%dimethylacetamide (DMAC) in acidified saline via IV bolus for rat and 5min or 10 min IV infusion for monkey. For oral (PO) dosing, testcompounds are dosed at 1.0 to 3.0 mg/kg using 5% DMAC in 0.5%methylcellulose in citrate buffer (pH 2.5). Blood samples are collectedat predose and various time points up to 24 hours postdose. All bloodsamples are collected using EDTA as the anticoagulant and centrifuged toobtain plasma samples. The plasma concentrations of test compounds aredetermined by LC-MS methods. The measured plasma concentrations are usedto calculate PK parameters by standard noncompartmental methods usingPhoenix® WinNonlin software program (version 8.0, PharsightCorporation).

In rats and monkeys, cassette dosing of test compounds are conducted toobtain preliminary PK parameters.

In vivo pharmacokinetic experiments with male beagle dogs may beperformed under the conditions described above.

Example L: Time Dependent Inhibition (TDI) of CYP Protocol

This assay is designed to characterize an increase in CYP inhibition asa test compounds is metabolized over time. Potential mechanisms for thisinclude the formation of a tight-binding, quasi-irreversible inhibitorymetabolite complex or the inactivation of P450 enzymes by covalentadduct formation of metabolites. While this experiment employs a 10-folddilution to diminish metabolite concentrations and therefore effects ofreversible inhibition, it is possible (but not common) that a metabolitethat is an extremely potent CYP inhibitor could result in a positiveresult.

The results are from a cocktail of CYP specific probe substrates at 4times their Km concentrations for CYP2C9, 2C19, 2D6 and 3A4 (midazolam)using human liver microsomes (HLM). The HLMs can be pre-incubated withtest compounds at a concentration 10 μM for 30 min in the presence (+N)or absence (−N) of a NADPH regenerating system, diluted 10-fold, andincubated for 8 min in the presence of the substrate cocktail with theaddition of a fresh aliquot of NADPH regenerating system. A calibrationcurve of metabolite standards can be used to quantitatively measure theenzyme activity using LC-MS/MS. In addition, incubations with known timedependent inhibitors, tienilic aicd (CYP2C9), ticlopidine (CYP2C19),paroxetine (CYP2D6), and troleandomycin (CYP3A4), used as positivecontrols are pre-incubated 30 min with or without a NADPH regeneratingsystem.

The analytical system consists of a Shimadzu LC-30AD binary pump systemand SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia,Md.) coupled with a Sciex Triple Quad 6500+ mass spectrometer fromApplied Biosystems (Foster City, Calif.). Chromatographic separation oftest compounds and internal standard can be achieved using an ACQUITYUPLC BEH 130A, 2.1×50 mm, 1.7 μm HPLC column (Waters Corp, Milford,Mass.). Mobile phase A consists of 0.1% formic acid in water, and mobilephase B consists of 0.1% formic acid in acetonitrile. The total LC-MS/MSruntime will be 2.50 minutes with a flow rate of 0.9 mL/min. Peak areaintegrations and peak area ratio calculations are performed usingAnalyst software (version 1.6.3) from Applied Biosystems.

The percentage of control CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activityremaining following preincubation of the compounds with NADPH iscorrected for the corresponding control vehicle activity and thencalculated based on 0 minutes as 100%. A linear regression plot of thenatural log of % activity remaining versus time for each isozyme is usedto calculate the slope. The −slope is equal to the rate of enzyme loss,or the K_(obs).

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including withoutlimitation all patent, patent applications, and publications, cited inthe present application is incorporated herein by reference in itsentirety.

1. A compound having Formula I:

or a pharmaceutically acceptable salt thereof, wherein: Y is N or OH; R¹is selected from Cl, CH₃, CH₂F, CHF₂, and OF₃; Cy¹ is selected from

R² is selected from F and Cl; R³ is selected from

and, Cy² is selected from

provided that the compound of Formula I is other than,2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile,2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile, and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile.


2. The compound of claim 1, wherein the compound of Formula I is acompound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom Cl and CH₃; Cy¹ is selected from

R³ is selected from

and, Cy² is selected from


3. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein: Y is N or OH; R¹ is selected from Cl, CH₃, CH₂F, CHF₂,and OF₃; Cy¹ is selected from

R² is selected from F and Cl; R is selected from

and, Cy² is selected from

provided that the compound of Formula I is other than2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile.


4. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Y is N or OH; R¹ is selected from Cl, CH₃, CH₂F, CHF₂,and OF₃; Cy¹ is selected from

R² is selected from F and Cl; R³ is selected from

and, Cy² is selected from


5. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Y is CH.
 6. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein Y is N.
 7. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Cy¹ is selected from Cy¹-a, Cy¹-b, Cy¹-c, Cy¹-d, and Cy¹-e. 8.The compound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Cy¹ is selected from Cy¹-f, Cy¹-g, Cy¹-h, Cy¹-i, Cy¹-j, andCy¹-k.
 9. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Cy¹ is selected from Cy¹-a and Cy¹-b.
 10. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein Cy¹is selected from Cy¹-1, Cy¹-m, Cy¹-n, Cy¹-o, Cy¹-p, Cy¹-q, Cy¹-r, Cy¹-s,and Cy¹-t.
 11. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein Cy¹ is selected from Cy¹-c, Cy¹-m, Cy¹-n, Cy¹-o,Cy¹-p, Cy¹-q, Cy¹-r, Cy¹-s, and Cy¹-t.
 12. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is selected fromCH₃, CH₂F, CHF₂, and CF₃.
 13. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is selected fromCH₃ and CF₃.
 14. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R² is F.
 15. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein R² is Cl.
 16. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R³ is selected from R³-a and R³-b.
 17. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein R³ is selectedfrom R³-b and R³-c.
 18. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein Cy² is selected from Cy²-a, Cy²-b, andCy²-d.
 19. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein Cy² is selected from Cy²-a and Cy²-b.
 20. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Cy² is selected from Cy²-b and Cy²-d.
 21. The compound of claim1, wherein the compound of Formula I is selected from2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(2-methoxy-3-methylphenyl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(7-(3-chloro-2-methoxyphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;1-(4-(6-fluoro-7-(5-fluoroquinolin-8-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)piperidin-1-yl)prop-2-en-1-one;2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(2,3-dimethylphenyl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-6-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(6-methylpyridin-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methyl-1H-indazol-3-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(4-fluorophenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;8-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(7-(2-chloro-3-methylphenyl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-imidazo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;8-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;8-(6-fluoro-1-(1-((E)-4-fluorobut-2-enoyl)piperidin-4-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;8-(1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-naphthonitrile;2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6,8-dichloro-4-(3-(dimethylamino)-3-methylazetidin-1-yl)-7-(5-fluoroquinolin-8-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-fluorobut-2-enoyl)piperidin-2-yl)acetonitrile;and2-((2S,4S)-4-(4-(3-(dimethylamino)-3-methylazetidin-1-yl)-6-fluoro-7-(5-fluoroquinolin-8-yl)-8-(trifluoromethyl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;or a pharmaceutically acceptable salt thereof.
 22. The compound of claim1, wherein the compound of Formula I is selected from:2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(4-fluoroisoquinolin-1-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(3-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(7-fluoro-2-methylquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-8-methyl-7-(1-methylisoquinolin-4-yl)-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-((E)-4-methoxybut-2-enoyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(7-fluoroquinolin-8-yl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indazol-3-yl)acetonitrile;2-((2S,4S)-4-(4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-8-methyl-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-(4-(1-((2R,4S)-1-acryloyl-2-methylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile;2-(4-(1-(1-acryloylpiperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-7-yl)-1-methyl-1H-indol-3-yl)acetonitrile;2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;2-((2S,4S)-4-(6-fluoro-7-(2-fluoro-6-methoxyphenyl)-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;and2-((2S,4S)-4-(8-chloro-4-(3-(ethyl(methyl)amino)-3-methylazetidin-1-yl)-6-fluoro-7-(6-fluoro-5-methyl-1H-indazol-4-yl)-1H-[1,2,3]triazolo[4,5-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile;or a pharmaceutically acceptable salt thereof.
 23. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.
 24. A method of inhibiting KRAS activity, saidmethod comprising contacting a compound of claim 1, with KRAS.
 25. Themethod of claim 24, wherein the contacting comprises administering thecompound to a patient.
 26. A method of treating a disease or disorderassociated with inhibition of KRAS interaction, said method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of claim
 1. 27. A method of treating a disease ordisorder associated with inhibiting a KRAS protein harboring a G12Cmutation, said method comprising administering to a patient in needthereof a therapeutically effective amount of a compound of claim
 1. 28.A method for treating a cancer in a patient, said method comprisingadministering to the patient a therapeutically effective amount of thecompound of claim
 1. 29. The method of claim 28, wherein the cancer isselected from carcinomas, hematological cancers, sarcomas, andglioblastoma.
 30. The method of claim 29, wherein the hematologicalcancer is selected from myeloproliferative neoplasms, myelodysplasticsyndrome, chronic and juvenile myelomonocytic leukemia, acute myeloidleukemia, acute lymphocytic leukemia, and multiple myeloma.
 31. Themethod of claim 29, wherein the carcinomas is selected from pancreatic,colorectal, lung, bladder, gastric, esophageal, breast, head and neck,cervical, skin, and thyroid.
 32. The method of claim 26, wherein thedisease or disorder is an immunological or inflammatory disorder. 33.The method of claim 31, wherein the immunological or inflammatorydisorder is Ras-associated lymphoproliferative disorder and juvenilemyelomonocytic leukemia caused by somatic mutations of KRAS.